Apparatus for preparing a solution of a hyperpolarized noble gas for NMR and MRI analysis

ABSTRACT

The present invention relates generally to nuclear magnetic resonance (NMR) techniques for both spectroscopy and imaging. More particularly, the present invention relates to methods in which hyperpolarized noble gases (e.g., Xe and He) are used to enhance and improve NMR and MRI. Additionally, the hyperpolarized gas solutions of the invention are useful both in vitro and in vivo to study the dynamics or structure of a system. When used with biological systems, either in vivo or in vitro, it is within the scope of the invention to target the hyperpolarized gas and deliver it to specific regions within the system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/164,324filed on Jun. 5, 2002, now U.S. Pat. No. 6,818,202, which is adivisional of U.S. application Ser. No. 08/825,475 filed on Mar. 28,1997, now U.S. Pat. No. 6,426,058, which claims priority from U.S.provisional application Ser. No. 60/014,321 filed on Mar. 29, 1996.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Contract No.DE-AC03-765F00098, awarded by the Department of Energy. The Governmenthas certain rights in this invention.

REFERENCE TO A COMPUTER PROGRAM APPENDIX

Not Applicable

FIELD OF THE INVENTION

The present invention relates generally to nuclear magnetic resonance(NMR) techniques for both spectroscopy and imaging. More particularly,the present invention relates to the use of hyperpolarized noble gases(e.g., Xe and He) to enhance and improve NMR and MRI.

BACKGROUND OF THE INVENTION

Nuclear magnetic resonance (NMR) is an established technique for bothspectroscopy and imaging. NMR spectroscopy is one of the most powerfulmethods available for determining primary structure, conformation andlocal dynamic properties of molecules in liquid, solid and even gasphases. As a whole-body imaging technique, Magnetic Resonance Imaging(MRI) affords images possessing such superb soft tissue resolution thatMRI is the modality of choice in many clinical settings. MRI can produceimages which allow the clinician to distinguish between a pathologicalcondition and healthy tissue. For example, MR images clearlydifferentiate tumors from the surrounding tissue. Further, using MRI itis possible to image specific regions within the organism and to obtainanatomical (morphology and pathology) and/or functional informationabout various processes including blood flow and tissue perfusion.Functional imaging of the brain is now also well documented.

The structural and functional information available through MRI iscomplemented by whole-body NMR spectroscopy. NMR spectroscopic studieson organisms provides a means to probe the chemical processes occurringin the tissue under study. For example, the location and quantity ofintrinsic NMR spectroscopic markers such as lactate and citrate can bestudied to gain insight into the chemical processes underlying a diseasestate (Kurhanewicz, J., et al., Urology 45: 459-466 (1995)). NMRspectroscopy can also be used to observe the effects of administereddrugs on the biochemistry of the organism or the changes in the drugwhich occur following its administration (Maxwell, R. J., Cancer Surv.17: 415-423 (1993)). Efforts to improve the information yield from MRIand NMR spectroscopy through increased sensitivity or the use ofappropriately designed extrinsic probes have been ongoing since theinception of these techniques.

Sensitivity poses a persistent challenge to the use of NMR, both inimaging and spectroscopy. In proton MRI, contrast is primarily governedby the quantity of protons in a tissue and the intrinsic relaxationtimes of those protons (i.e., T₁ and T₂). Adjacent tissues which arehistologically distinct yet magnetically similar appear isointense on anMR image. As the proton content of a tissue is not a readily manipulableparameter, the approach taken to provide distinction betweenmagnetically similar tissues is the introduction into the biologicalsystem of a paramagnetic pharmaceutical (i.e, contrast enhancing agent)such as Gd(DTPA) (Niendorf, H. P., et al., Eur. J. Radiol., 13: 15(1991)). Interaction between the proton nuclei and the unpaired spins onthe Gd⁺³ ion dramatically decrease the proton relaxation times causingan increase in tissue intensity at the site of interaction. Gd(DTPA) andanalogous agents are small molecular agents which remain largelyconfined to the extracellular compartment and do not readily cross theintact blood-brain barrier. Thus, these agents are of little use infunctional brain imaging.

Similar to MRI, NMR spectroscopic studies generally rely on detectingNMR active nuclei which are present in their natural abundance (e.g.,¹H, ³¹P, ¹³C) (Sapega, A. A., et al., Med. Sci. Sports Exerc., 25:656-666 (1993)). Additionally, the chemical species under observationmust be spectroscopically distinguishable from the other compoundswithin the window of observation. Thus, sensitivity in NMR spectroscopyis a function of both the abundance and the spectral characteristics ofthe molecule(s) desired to be studied. The range of NMR spectroscopicstudies has been somewhat expanded by the application of exogenousprobes which contain NMR active nuclei, for example ¹⁹F (Aiken, N. R.,et al., Biochim. Biophys. Acta, 1270: 52-57 (1995)).

Noble gases are of interest as tracers and probes for MRI and NMRspectroscopy (Middleton, H., et al., Magn. Res. Med. 33: 271 (1995)),however, the sensitivity of MRI and NMR spectroscopy for these moleculesis relatively low. A factor which contributes to the lack of sensitivityof these techniques for the noble gases is that the spin polarization,or net magnetic moment, of the noble gas sample is low. For example, atypical molecule at thermal equilibrium at room tem has an excess ofspins in one direction along an imposed magnetic field relative to thosein the opposite direction of generally less than 1 in 10⁵. Lowertemperatures and higher fields, to the extent that these can be imposed,provide only limited benefit. Alternative approaches rely on disruptingthe equilibrium magnetization by forcing molecules in the sample into apolarized state. Two methods known in the art for enhancing the spinpolarization of a population of nuclei are dynamic nuclear polarizationand optical pumping.

Dynamic nuclear polarization, originally applied to metals, arises fromthe cross relaxation between coupled spins. The phenomenon is known asthe Overhauser Effect with early disclosures by Overhauser and others(Ovehauser, A. W., “Polarization of nuclei in metals,” Phys. Rev. 92(2):411-415 (1953), Solomon, I., “Relaxation processes in a system of twoSpins,” Phys. Rev. 99(2): 559-565 (1955), and Carver, T. R., et al.,“Experimental verification of the Overhauser nuclear polarizationeffect,” Phys. Rev. 102(4): 975-980 (1956)). The Nuclear OverhauserEffect between nuclear spins is widely used to determine interatomicdistances in NMR studies of molecules in solution.

Optical pumping is a method for enhancing the spin polarization of gaseswhich consists of irradiating an alkali metal, in the presence of anoble gas, with circularly polarized light. The hyperpolarized gasesthat result have been used for NMR studies of surfaces and imaging voidspaces and surfaces. Examples are the enhanced surface NMR ofhyperpolarized ¹²⁹Xe, as described by Raftery, D., et al., Phys. Rev.Lett. 66: 584 (1991); signal enhancement of proton and ¹³C NMR bythermal mixing from hyperpolarized ¹²⁹Xe, as described by Driehuys, B.,et al., Phys. Lett. A184: 88-92 (1993), and Bowers, C. R., et al., Chem.Phys. Lett. 205: 168 (1993), and by Hartmann-Hahn cross-polarization, asdescribed by Long, H. W., et al., J. Am. Chem. Soc. 115: 8491 (1993);and enhanced MRI of void spaces in organisms (such as the lung) andother materials, as described by Albert, M. S., et al., Nature 370:199-201 (1994), and Song, Y.-Q., et al., J. Magn. Reson. A115: 127-130(1995).

Although hyperpolarize noble gases have proven useful as probes in thestudy of the air spaces in lungs, the effectiveness or sensitivity ofthese methods is somewhat compromised for biological materials andorgans, such as blood and the parts of the body that are accessible onlythrough the blood. During its residence time in the blood, thehyperpolarized gas is diluted considerably and the delay in transferringthe gas from the lung space to the blood consumes much of the time(e.g., T₁) required for the polarized gas to return to itsnon-hyperpolarized state. Further complicating the situation, thepenetration of the hyperpolarized gas into the interior of red bloodcells dramatically reduces the T₁ of the hyperpolarized gas and thus,sorely attenuates the temporal range over which the gas can serve as aneffective probe.

A considerable advance in both MRI and NMR spectroscopy could beachieved by the introduction of a versatile hyperpolarized noblegas-based NMR active tracer which could also function as a contrastenhancing agent or otherwise affect, in a spectroscopically discernablemanner, sample molecules to which the probe is proximate. Among otherapplications, such an agent would be useful in conjunction withfunctional imaging of the brain and also to probe the dynamics ofexchange between the intracellular and extracellular compartments ofvarious tissues. Of even more profound significance would be a means ofdelivering the tracer, either through the blood or via direct injectioninto the tissue of interest, which maintains the hyperpolarization ofthe gas during the delivery process and through the imaging orspectroscopic experiment. Quite surprisingly, the instant inventionprovides both such a tracer and delivery method.

SUMMARY OF THE INVENTION

The present invention provides methods for using hyperpolarized noblegases in conjunction with NMR spectroscopy and MRI. The noble gases areuseful both as tracers, which are themselves detected, and also asagents which affect the magnetic properties of other nuclei present in asample.

Thus, in a first aspect, the present invention provides a method foranalyzing a sample containing an NMR active nucleus, the methodcomprising:

-   -   (a) contacting the sample with a hyperpolarized noble gas;    -   (b) scanning the sample by nuclear magnetic resonance        spectroscopy, magnetic resonance imaging, or both nuclear        magnetic resonance spectroscopy and magnetic resonance imaging;    -   (c) detecting the NMR active nucleus, wherein the NMR active        nucleus is a nucleus other than a noble gas.

In another aspect, the present invention provides a method for analyzinga sample which comprises: (a) combining a hyperpolarized noble gas witha fluid to form a mixture; (b) contacting the sample with the mixture;and (c) scanning the sample, the noble gas or both the sample and thenoble gas by nuclear magnetic resonance spectroscopy, magnetic resonanceimaging, or both nuclear magnetic resonance spectroscopy and magneticresonance imaging.

In a further aspect, the invention provides a pharmaceutical compositionwhich comprises a hyperpolarized noble gas dissolved in aphysiologically compatible liquid carrier.

In yet another aspect, the present invention provides a method forstudying a property of a noble gas in a tissue. This method of theinvention comprises: (a) hyperpolarizing a noble gas; (b) dissolving thehyperpolarized noble gas in a physiologically compatible liquid carrierto form a mixture; (c) contacting the tissue with the mixture from (b);and (d) scanning the tissue by nuclear magnetic resonance, magneticresonance imaging, or both, whereby the property of the noble gas in thetissue is studied.

In a further aspect, the invention provides a method for enhancing therelaxation time of a hyperpolarized noble gas in contact with aphysiological fluid. This method comprises: (a) forming a hyperpolarizednoble gas intermediate solution by dissolving the hyperpolarized noblegas in a fluid in which the relaxation time of the noble gas is longerthan the relaxation time of the noble gas in the physiological fluid;and (b) contacting the physiological fluid with the intermediatesolution.

In yet a further aspect, the present invention provides a method formeasuring a signal transferred from at least one hyperpolarized noblegas atom to at least one non-noble gas NMR active nucleus, comprising:(a) contacting a non-noble gas NMR active nucleus with a hyperpolarizednoble gas atom; (b) applying radiofrequency energy to the non-noble gasNMR active nucleus in a magnetic field; and (c) measuring the signaltransferred from the hyperpolarized noble gas atom to the non-noble gasNMR active nucleus using nuclear magnetic resonance spectroscopy,magnetic resonance imaging, or both.

In a still further aspect, the invention provides a pulse sequence forheteronuclear difference spin polarization induced nuclear Overhausereffect (SPINOE) NMR of a system comprising at least one hyperpolarizednoble gas atom and at least one non-noble gas NMR active nucleus,comprising: (a) at least one non-noble gas NMR active nucleus π/2 pulse;(b) a non-noble gas NMR active nucleus π pulse applied simultaneouslywith application of a noble gas π pulse; and (c) a non-noble gas NMRactive nucleus π/2 pulse.

In an additional aspect, the invention provides an apparatus forpreparing a solution of a hyperpolarized noble gas in a fluid, theapparatus comprising:

-   -   a vessel for receiving the fluid;    -   a reservoir for receiving the hyperpolarized noble gas, the        reservoir communicating through a first shutoff valve with the        vessel, the reservoir being shaped to allow the reservoir to be        cooled independently of the vessel;    -   a gas inlet port communicating through a second shutoff valve        with the reservoir; and    -   a means for withdrawing the fluid from the vessel independently        of the first and second shutoff valve.

Other features, objects and advantages of the invention and itspreferred embodiments will become apparent from the detailed descriptionwhich follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic is set forth of the experimental protocol used.Eighty percent of isotopically enriched ¹²⁹Xe is polarized via spinexchange with optically pumped rubidium atoms using previously describedtechniques. The xenon is frozen at liquid nitrogen temperature in asidearm of a sample tube in high magnetic field provided by a permanentmagnet. The xenon is then brought to the gas phase by warming andadmitted to the solution.

FIG. 2. ¹²⁹Xe NMR spectrum of a solution of ¹²⁹Xe in D₂O

FIGS. 3A and 3B. Conventional and optically polarized ¹²⁹Xe NMR spectraof xenon in blood acquired after injecting 1 cc of xenon/water mixtureinto 1 cc of concentrated red blood cells are set forth.

FIG. 4. The time dependence of the integrals of the two peaks in atypical ¹²⁹NMR of ¹²⁹Xe in blood is set forth.

FIGS. 5A and 5B. Intrinsic exchange of xenon between the extracellularand intracellular compartments of blood. FIG. 5A shows the initialequilibrium spectrum and the time dependent spectra following theselective inversion. FIG. 5B shows the time dependence of the signalintensities.

FIGS. 6A and 6B. Optically pumped ¹²⁹Xe spectrum of xenon delivered toblood in the INTRALIPID® solution is provided (A). 2-dimensional ¹²⁹XeMR image laser-polarized xenon in blood/INTRALIPID® (A, inset). A ¹²⁹Xespectrum acquired after mixing the xenon/FLUOSOL® solution in wholeblood (B). Compressed ¹²⁹Xe NMR spectrum of xenon/FLUOSOL® solution inwhole blood (B, inset).

FIG. 7. Two-dimensional magnetic resonance image of ¹²⁹Xe dissolved infresh human blood taken immediately after the blood is mixed with thesaline saturated by hyperpolarized xenon. The 128×64 images were takenby the Echo Planar Imaging (EPI) method on a Quest 4300 spectrometer.The diameter of the sample tube is 10 mm, and the solution occupies aregion of length of 20 mm.

FIG. 8. Time dependence of the hyperpolarized ¹²⁹Xe NMR signal observedin benzene solution after being contacted with hyperpolarized xenon. Themain figure shows the data for partially deuterated benzene (25% C₆D₅H,75% C₆D₆); the inset shows the data for normal benzene (C₆H₆). In theexperiments represented by open circles, xenon was admitted into benzeneby opening the xenon reservoir; the initial rise in signal representsthe penetration of xenon into the solvent. In the experiment representedby closed circles, the xenon was mixed with the benzene by shaking thesample after opening the xenon reservoir, so as to produce a uniformsaturated solution. ¹²⁹Xe spin polarization was enhanced by opticalpumping using circularly polarized light at 794.7 nm. Typically, 4×10⁻⁴moles of enriched ¹²⁹Xe were used in one experiment. The difference inthe ¹²⁹Xe signal between benzene and deuterated benzene demonstrates theeffect of magnetic dipolar coupling between ¹H and ¹²⁹Xe spins on therelaxation of the ¹²⁹Xe. For the initial NOE experiments, the partiallydeuterated liquids were used in order to favor the effects ofcross-relaxation over those contributing to ¹H auto-relaxation. ¹²⁹XeNMR was performed at 51 MHz on a Quest 4300 spectrometer using ahome-built probe and a tipping angle of 3°.

FIG. 9. Time dependence of the ¹H NMR signal observed after exposure ofpartially deuterated benzene (25% C₆D5H, 75% C₆D₆) to hyperpolarized¹²⁹Xe. The sample was exposed to xenon at zero magnetic field and wasthen inserted into the NMR probe within a few seconds. The initial riseof the ¹H signal is due to spin-lattice relaxation. The ¹H NMR signalexhibits a positive (◯) or negative (⋄) NOE depending on the sign of the¹²⁹Xe polarization. From the variation of the ¹H signal in the presenceof unpolarized xenon (□), the ¹H T₁ of the benzene-xenon solution isdetermined to be ˜160 s. Inset: Time dependence of the ¹H NMR signalafter polarized ¹²⁹Xe was dissolved in partially deuterated benzene.Prior to admitting the xenon, the sample was placed in the NMR magnetfor approximately 10 minutes to allow thermal equilibration of the ¹Hmagnetization. After the xenon reservoir was opened, the sample was thenshaken to ensure efficient mixing of the xenon and benzene. The smoothlines represent a fit to the time dependent solution (J. H. Noggle, R.E. Schirmer, The Nuclear Overhauser Effect: Chemical Applications(Academic Press, New York-London-Toronto-Sidney-San Francisco, 1971)) ofEq. 1.l(t)=a+b(e ^(−t/t) ¹ −e ^(−t/t) ² )  (1)yielding time constants of 120 s and 1050 s (●), and 140 s and 1020 s(♦). ¹H NMR was performed at 185 MHz using a home-built probe and atipping angle of 3°.

FIG. 10. Time-resolved, two-dimensional magnetic resonance images of¹²⁹Xe dissolved in benzene, taken after the exposure of the benzene tohyperpolarized ¹²⁹Xe. A Xe concentration gradient exists immediatelyafter the Xe is admitted, evolving with time to a more uniform solution.The 64 pixel by 128 pixel images were taken by the fast low-angle shot(FLASH) imaging method on a Quest 4300 spectrometer, with a tippingangle of 3° for each of the 64 signal acquisitions. Thefrequency-encoding gradient was 3.5 G/mm. The step size of thephase-encoding gradient pulses, which were 500 μs long, was 0.063 G/mm.The diameter of the sample tube is 7 mm, and the solution occupies aregion of length 15 mm.

FIG. 11. Time-resolved distribution of ¹²⁹Xe magnetization in partiallydeuterated benzene from MRI projection along the tube axis (z). Thesample was not shaken after xenon was admitted to the benzene in orderto prevent a uniform initial concentration. In the first image taken 47s after the admission of the xenon gas to the solution three regions maybe distinguished. The intensity above the solution level (above 18 mm)arises from ¹²⁹Xe in the gas phase which is displaced from the dissolved¹²⁹Xe signal due to its different chemical shift. The decrease of thegas signal above 21 mm along the z axis is due to the declining NMRsensitivity beyond the radiofrequency coil, represented by circles inthe schematic. The signal maximum at a position of 15.2 mm correspondsto the top of the solution, arising from xenon diffusing into thesolution from the gas phase. The signal maximum at about 1.3 mmcorresponds to the lower end of the tube. Thus, xenon accumulates at thebottom of the sample tube first and a discernible xenon concentrationgradient persists for up to 5 minutes. The concentration gradientresults from natural convection due to density differences between thexenon solution and that of pure benzene, progressing ultimately to auniform saturated xenon solution. The imaging field gradient was 2.6G/mm.

FIG. 12. Two-dimensional magnetic resonance images of the NOE enhanced¹H signals at 2 and 6 minutes after hyperpolarized xenon was admitted tothe sample tube containing normal benzene. The enhancement images wereobtained by subtracting the equilibrium image shown, which is theaverage of four images taken after 25 minutes. The intensity scale inthe difference images has been magnified 8-fold for clarity. The maximumNOE enhancement in the 2 minute image is 0.05; that in the 6 minuteimage is 0.12. A perceptible gradient of the enhanced ¹H signal isobserved in the 2 minute image, corresponding to the observed gradientin the xenon concentration and the enhancement is found to be uniform inthe 6 minute image when the xenon concentration gradient is diminished.The negative region in the 2 minute image could be caused by expansionof the liquid phase as xenon dissolves. The images were taken by theEcho Planar Imaging method (Mansfield, P., J. Phys. C 10, L55 (1977)) in24 ms. The frequency-encoding gradient was 3.15 G/mm; the phase-encodinggradient pulses were 0.14 G/mm and 50 μs long. The image dimension was128×32, and the image was zero-filled to 256×256 in data processing. Theskew of the image is due to the inhomogeneity of the static magneticfield.

FIG. 13. Schematic diagram of the pulse sequence used to obtainheteronuclear difference SPINOE spectra. The proton magnetization issaturated first by a series of π/2 pulses and a z-axis magnetic fieldgradient is applied in between the pulses to dephase the transversecomponents of the magnetization for optimal saturation. The π pulseshelps to reduce the growth of proton signal due to spin-latticerelaxations. A π pulse is also applied to the ¹²⁹Xe resonance at thesame time of the proton π pulses so that the ¹²⁹Xe magnetization isinverted in synchronization with the proton magnetization. Thissynchronization ensures that the SPINOE signals will be accumulatedduring the entire mixing time. Both proton and xenon π pulses areadiabatic pulses BIR4 of 1 ms in duration.

FIGS. 14A and 14B. (A) Proton spectra of 0.1 M p-nitrotoluene solutionin perdeuterated benzene at thermal equilibrium; (B) SPINOE protonspectra of 0.1 M p-nitrotoluene solution in perdeuterated benzene withpositive and negative ¹²⁹Xe spin polarization. The total mixing time is2.1 s.

FIG. 15. Proton spectra of 0.05 M α-cyclodextrin solution inperdeuterated DMSO (dimethyl sulfoxide) at thermal equilibrium;

FIG. 16. SPINOE spectrum of α-cyclodextrin in the presence of negativelypolarized ¹²⁹Xe.

FIG. 17. SPINOE spectrum of α-cyclodextrin in the presence of positivelypolarized ¹²⁹Xe. The positive ¹²⁹Xe polarization is defined to be alongthe thermal equlibrium polarization. The total mixing time is 1 s andtwo signals were acquired for each spectrum.

FIG. 18. Schematic diagram showing the process used for in vivo imagingof hyperpolarized 129Xe in the rat.

FIG. 19. A ¹²⁹Xe xenon spectrum representing an average of the sixththrough the twelfth scan in a series of ¹²⁹Xe spectra taken over thethorax and abdomen areas following intravenous injection of axenon/INTRALIPID® solution in the rat.

FIG. 20. Schematic diagram of the ¹²⁹Xe imaging experiment showing thetiming of and relationship between the excitation pulse, slice selectionpulse, first and second gradients and signal detection.

FIG. 21. Two dimensional ¹²⁹Xe images taken at intervals ofapproximately 7 seconds. The images depict the ¹²⁹Xe signal intensity inthe upper part of the rat's hind leg.

FIG. 22. A representation of one possible apparatus to accomplish themixing of a hyperpolarized noble gas with a fluid as contemplated bythis invention. The apparatus has four main subcomponents: a vessel forreceiving the fluid 10, a noble gas reservoir 20, a gas inlet port 40,and a means to remove the liquid from the vessel 60. The reservoir andthe vessel are connected by means of a shutoff valve 30. Similarly, thereservoir and the gas inlet port are connected via a shutoff valve 50.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

It has been discovered that when a hyperpolarized noble gas (e.g.,¹²⁹Xe) is dissolved in liquid solvents, a time dependent departure of,e.g., the proton spin polarization from its thermal equilibrium isobserved. The variation of the magnetization, positive or negativedepending on the sign of the spin polarization of the noble gas, is anunexpected manifestation of the nuclear Overhauser effect (NOE), aconsequence of cross relaxation between the spins of the solutionprotons and the dissolved hyperpolarized noble gas. Time-resolvedmagnetic resonance images of both nuclei, ¹H and dissolved noble gas, insolution show that the proton magnetization is selectively perked inregions containing the spin-polarized noble gas. Thus, it has now beendetermined that optical pumping and the nuclear Overhauser effect caneffectively be used to transfer enhanced polarization fromhyperpolarized noble gas to solution phase species without requiring theneed for radiofrequency irradiation of the perturbing spins, an effectwhich is denoted Spin Polarization Induced Nuclear Overhauser Effect(SPINOE). Thus, SPINOE can advantageously be used to enhance thesensitivity of NMR and, in turn, to better determine the primarystructure, conformation and local dynamic properties of the molecules ina liquid solution.

As such, in one aspect, the present invention provides a method foranalyzing a sample containing an NMR active nucleus. This methodcomprises:

(a) contacting the sample with a hyperpolarized noble gas; (b) scanningthe sample using nuclear magnetic resonance spectroscopy, magneticresonance imaging, or both nuclear magnetic resonance spectroscopy andmagnetic resonance imaging; and (c) detecting the NMR active nucleus,wherein the NMR active nucleus is a nucleus other than a noble gas.

The term “contacting” is used herein interchangeably with the following:combined with, added to, dissolved in, mixed with, passed over, flowedover, administered to, injected into, ingested by, etc. The sample canbe contacted with the hyperpolarized noble gas in a liquid, solid or gasphase. Further, the sample studied may be a liquid, solid, a combinationof a liquid and a solid or the boundary between a solid and a liquid.Prior to contacting the sample with the hyperpolarized noble gas, it maybe desirable to freeze the noble gas to preserve the hyperpolarization.Further, freezing the gas in a magnetic field can preserve thehyperpolarization for a period which is significantly longer than thatobtained simply by freezing the gas. For those noble gases which freezeat temperatures which are difficult to achieve, it is within the scopeof this invention to cool those gases to a temperature above theirfreezing point. This procedure is encompassed by the term “freezing.”Similar to that described above, such cooling can also occur in thepresence of a magnetic field

Once contacted with the noble gas, the sample can be scanned using NMR,MRI or both. The sample is scanned to detect the effects of thehyperpolarized gas on NMR active nuclei within the sample. Any non-noblegas NMR active nucleus can be detected. As used herein, “NMR activenucleus” denotes those nuclei which have a nonzero spin quantum number.Such NMR active nuclei include, but are not limited to, ¹H, ¹³C, ¹⁵N,¹⁹F, ²⁹Si, ³¹P and combinations thereof. In preferred embodiments,multiple NMR active nuclei are detected. By detecting the effects of thehyperpolarized noble gas on the sample, one can readily analyze thestructure, chemistry, spatial distribution, etc. of the sample.

In another aspect, the present invention provides a method for analyzinga sample which is based on the discovery that a noble gas can becombined with a fluid to form a mixture and, in turn, the mixture can bedelivered to blood or other tissue while the noble gas still has a largeoff-equilibrium nuclear spin polarization. Thus, this method comprises:(a) combining a hyperpolarized noble gas with a fluid to form a mixture;(b) contacting the sample with the mixture; and (c) scanning the sample,the noble gas or both the sample and the noble gas by nuclear magneticresonance spectroscopy, magnetic resonance imaging or both nuclearmagnetic resonance spectroscopy and magnetic resonance imaging.

As used herein, the term “fluid” includes, but is not limited to water,saline, phosphate buffered saline, aqueous bufffer solutions,fluorocarbons, fluorocarbon solutions in water or organic solvents,aqueous fluorocarbon emulsions, lipids, solutions of lipids organicsolvents, aqueous emulsions of lipids, organic solvents (e.g., DMSO,ethanol, etc.). “Aqueous” encompasses solutions and emulsions preparedwith ¹H₂O, ²H₂O or ³H₂O. The terms “fluid,” “liquid” and “liquidcarrier” are used interchangeably herein.

In preferred embodiments, the noble gas is selected from the groupconsisting of xenon, helium, neon, krypton and mixtures of these gases.In more preferred embodiments, the noble gas is xenon and inparticularly preferred embodiments, the noble gas is either ¹²⁹Xe or¹³¹Xe. In this method, it is desirable to pre-dissolve thehyperpolarized noble gas in a fluid which can, for example, prolong itsrelaxation time when the hyperpolarized xenon is in contact withphysiological fluids. For instance, if the hyperpolarized gas is to beinjected into blood, it is desirable to first pre-dissolve thehyperpolarized gas in a lipid, lipid solution or lipid emulsion to forma mixture which, in turn, is injected into the blood. Also desirable isdissolving the hyperpolarized noble gas in a fluorocarbon, fluorocarbonsolution or fluorocarbon emulsion. The means of making such lipid andfluorocarbon formulations will be apparent to those of skill in the art.Moreover, it may be desirable to use a hyperpolarized noble gas topolarize a fluid which, in turn, is used as the contrasting agent orprobe. For instance, it may be desirable to polarize water by combiningit with a hyperpolarized noble gas and, thereafter, use the polarizedwater as the contrasting agent or probe. It may also prove advantageousto dissolve the noble gas in a liquid prior to hyperpolarizing the noblegas.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a hyperpolarized noble gas dissolved in aphysiologically compatible liquid carrier. In preferred embodiments, theliquid carrier is compatible with administration of the hyperpolarizedgas by percutaneous, intravenous, oral, intraperitoneal, intramuscularor inhalation routes. In certain more preferred embodiments, the liquidcarrier is appropriate for administration to an organism via anintravenous route.

As noted above, the hyperpolarized noble gas is combined with a fluid orliquid carrier which is chemically, biologically or materiallycompatible with the sample to be analyzed or, in some instance,dissolves as much of the noble gas as possible. Fluids suitable for usein the methods of the present invention include, but are not limited to,water, saline water, isotonic buffers, lipids, lipid emulsions, organicsolvents, fluorocarbon blood substitutes and other medically safeintravenous or oral media in which the noble gas relaxation time issufficiently long.

In preferred embodiments, the fluid in which the noble gas is dissolvedis a fluorocarbon or aqueous perfluorocarbon emulsion. Preferred speciesare perfluorocarbons including, but not limited to, perfluorodecalin,perfluoro-1,3-dimethylcyclohexane, perfluorohexane(s), perfluorohexyliodide, perfluoro(methylcyclohexane), perfluoro(methyldecalin),perfluoro-2-methyl-2-pentene, perfluorononane, perfluorooctane(s),perfluorobutylamine and perfluorotriethylamine. The only caveat to theuse of perfluorcarbons is that, where it is desired to use fluorocarbonsin vivo, the fluorocarbons must be compatible with the biological systemunder study. Those of skill in the art will readily be able to discernwhether the fluorocarbon is compatible with the biological system. Forin vitro applications, such compatibility is desirable but is notessential.

Particularly preferred fluorocarbons are those known in the art to besafe for in vivo administration. Of those safe for in vivoadministration, perfluorocarbons which are useful as blood substitutesare the most preferred. Perfluorocarbons useful as blood substitutes areknown in the art. (See, for example, Long, D. M., et al. in BLOODSUBSTITUTES, Chang, T. M. S. and Geyer, R. D., Eds. Marcel Dekker, Inc.New York, 1989, pp 411-420, which is herein incorporated by reference.).Examples of perfluorocarbons used as blood substitutes includeperfluorooctylbromide (PFOB), perfluortributylamine andperfluorodecalin. Fluorocarbons can be used as neat liquids, emulsions,or they can be dissolved in a solvent or injection adjuvant prior totheir use.

Fluorocarbon emulsions can be formed with water, plasma, blood, buffersor other aqueous constituents. Methods of producing pharmaceuticallyacceptable solutions and emulsions are well known to those of skill inthe art and any means known in the art for preparing these mixtures canbe used to practice the instant invention. (See, Naim, J. G., inREMINGTON'S PHARMACEUTICAL SCIENCES, Vol. 17, Gennaro, A. R., Ed., MackPublishing Co., Easton, Pa., 1985, pp. 1492-1517, which is incorporatedherein by reference.).

Fluids particularly preferred in practicing the present invention arecommercially available blood substitutes such as PFB-1, PFB-2 (AlliancePharmaceutical Corp.) and FLUOSOL®. FLUOSOL®, an intravascularperfluorocarbon emulsion which is commercially available from AlphaTherapeutic Corporation (Los Angeles, Calif., U.S.A.), is exemplary of afluorocarbon blood substitute which can be used in the methods of thepresent invention. Other fluorocarbons and fluorocarbon formulationsuseful in practicing the invention will be apparent to those of skill inthe art.

In another embodiment, the noble gas is dissolved in a lipid, lipidsolution or lipid emulsion. The term “lipid” refers to any oil or fattyacid derivative. The oil may be derived from vegetable, mineral oranimal sources. As used herein, the term “lipid” also includes thoselipids which are capable of forming a bilayer in aqueous medium, such tha hydrophobic portion of the lipid material orients toward the bilayerwhile a hydrophilic portion orients toward the aqueous phase.Hydrophilic characteristics derive from the presence of phosphato,carboxylic, sulfato, amino, sulfhydryl, nitro and other like groups.Hydrophobicity can be conferred by the inclusion of groups tat include,but are not limited to, long chain saturated and unsaturated aliphatichydrocarbon groups and such groups substituted by one or more aromatic,cycloaliphatic or heterocyclic group(s). Preferred lipids arephosphoglycerides and sphingolipids, representative examples of whichinclude phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lacking inphosphorus, such as sphingolipid and glycosphingolipid families, arealso within the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

Particularly preferred in practicing this embodiment of the presentinvention is the use of a commercially available lipid preparation suchas 10% or 20% INTRALIPID® (Clintec Nutrition, Deerfield, Ill., U.S.A.),or 10% or 20% LIPOSYN® II, or 10% or 20% LIPOSYN® III. LIPOSYN® is anintravenous fat emulsion which is commercially available from AbbotLaboratories (Abbot Park Ill., U.S.A.), and is exemplary of a lipidemulsion which can be used in the methods of the present invention.Lipid emulsions are particularly useful because they dissolve the noblegases and, in addition, because the noble gases have long relaxationtimes in such lipids. Other lipids, lipid mixtures and fluids in generalwhich are suitable for use in accordance with the present invention willbe apparent to those of skill in the art.

It should be noted that it is often desirable to add a deuterated orpartially deuterated solvent to the mixture. Moreover, intramuscularinjection adjuvants, such as DMSO, vitamin E, etc., can also be used ascarriers of the noble gas. Many of these fluids are readily availablefrom commercial sources. Other compounds which are solvents for noblegases and also have pharmaceutically acceptable or pharmacologicallyuseful properties will be apparent to those of skill in the art.

In certain preferred embodiments, the fluid into which the noble gas isdissolved will have the property of specifically or selectivelytargeting a specific organ or tissue within an organism. Many methods ofachieving such targeting are known in the art. For example, lipidvesicles (liposomes) are known to be rapidly scavenged by the cells ofthe reticuloendothelial system (RES). Thus, in one embodiment, polarizednoble gas is targeted to the RES by its incorporation into a liposome.Certain liposomes (“Stealth liposomes”) are known which avoid the cellsof the RES and remain primarily intravascular over their period of invivo residence. Thus, in another embodiment, the hyperpolarized noblegas is incorporated into a “Stealth liposome” and is used as anintravascular agent. Other liposomes for use in the present inventioninclude temperature sensitive liposomes, target-sensitive liposomes andpH-sensitive liposomes. Each of these liposomes is well known in theart. (See, Oku, N. LIPOSOMES, pp. 24-33, in POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, Dunn, R. L., et al., Eds. ACS Symposium Series 469,American Chemical Society, Washington, D.C., 1991, which is hereinincorporated by reference.).

The use of molecules which have a chemical avidity for receptors on cellsurfaces to deliver pharmaceutical agents to those cells is well knownin the art. It is within the scope of the instant invention to dissolvea noble gas in a fluid containing a molecule with avidity for specifictissues or cells and exploit this avidity to deliver the noble gas tothe tissue or cells. Each of the above-detailed embodiments can be usedboth in vitro and in vivo.

The above discussion regarding the use of liposomes andreceptor-mediated targeting of polarized noble gases is intended toserve as an example of methods and delivery vehicles which are useful inconjunction with the present invention. These examples are not intendedto define or limit the invention or the embodiments of the inventionwherein the noble gas is targeted to specific tissues.

Once formed, the noble gas/liquid mixture can be combined with thesample using a number of different techniques known to those of skill inthe art. For example, if the sample is a mammalian organism or a portionthereof, the mixture can be administered to the organism by, forexample, injection, inhalation or ingestion. More particularly,depending upon its intended use, the noble gas/liquid mixture can beinjected into the tissue of interest (if clinically harmless), orintravascularly to be delivered to the tissue of choice. In addition,the noble gas/liquid mixture can be swallowed or, alternatively, a noblegas/liquid aerosol can be inhaled for certain medical imagingapplications. Once the noble gas/liquid mixture has been administered tothe sample, the sample is scanned by nuclear magnetic resonance and/orby magnetic resonance imaging for purposes of molecular structuralstudies and/or spatial distribution. It should be noted that the noblegas/liquid mixture can be administered a single time or, alternatively,on a continuous or quasi-continuous basis.

As used herein the term “sample” encompasses diverse structures and caninclude an organism. “Sample” also include organic monomers andpolymers, inorganic monomers and polymers, biopolymers including, butnot limited to, oligopeptides, polypeptides, antibodies, proteins,oligonucleotides, polymers of ribonucleic acids (e.g., RNA, mRNA, tRNA)and deoxyribonucleic acids (DNA) including, but not limited to,chromosomes, genes and plasmids. Also encompassed within the term“sample” are carbohydrates, including oligosaccharides, polysaccharides,glycoproteins and mucopolysaccharides, lipids, blood, carbohydrates,catalysts, polymers, porous materials (e.g., surfaces, chemical reactorbeds, rocks present in oil reserves), etc. A “sample” can nativelycontain NMR active nuclei, NMR inactive nuclei or a combination of NMRactive and NMR inactive nuclei. Using the methods of the presentinvention, one can readily analyze the structure, chemistry, spatialdistribution, etc. of such samples. Other samples which can be analyzedusing the methods of the present invention will be readily apparent tothose of skill in the art.

As used herein, the term “organism” refers to life forms including, forexample, animals, plants, microorganisms and fungi. Methods of theinvention can be used with organisms which are either living or dead.The term “organism” also encompasses portions of organisms (e.g.,organs, organ group(s), tissue(s), etc.) either in situ or removed fromthe organism to which they are native.

The term “organ” refers to individual functional components of anorganisms including heart, liver, lungs, blood, brain, muscle, etc.“Organ group” as used herein, refers to cooperative organ systems, forexample, reticuloendothelial, central nervous, peripheral nervous,digestive, etc. As used herein “tissue” means a cell or an aggregate ofsimilar cells including, for example, blood, bone, muscle, nerve, etc.That some overlap exists between the structures encompassed by the terms“organ,” “organ group,” and “tissue” should be recognized; these termsare not intended to be mutually exclusive.

As used herein, the term “organic monomer” refers to a small organic(i.e., carbon containing) molecule with a molecular weight typicallyfalling within the range of from about 15 daltons to about 1000 daltons.Beyond a general adherence to the stated molecular weight range, nolimitation on the structure or functionality of these molecules isintended. This term encompasses both synthetic and natural compounds.Further, an “organic monomer” may also comprise one or more inorganicmolecules such as is found in, for example, organic chelates, chelatingresins, organometallic compounds and metalloporphyrins.

Complementary to the term “organic monomer,” and similar in definition,is the term “organic polymer” which encompasses organic molecules of amolecular weight greater than about 1000 daltons. Both synthetic andnatural compounds are defined by this term. Organic polymers may includematerials such as, for example, engineering plastics, textile polymersand polymers with medical applications.

As used herein, the term “inorganic monomer” refers to a small inorganicmolecule with a molecular weight typically falling within the range offrom about 1 dalton to about 1000 daltons. “Inorganic monomer”complements the term “organic monomer” and thus, encompasses moleculeswhich do not incorporate carbon as part of their structure.Complementary to the term “inorganic monomer” and similar in definitionis the term “inorganic polymer” which defines inorganic molecules of amolecular weight greater than 1000 daltons and encompasses bothsynthetic and natural polymeric materials.

The term “protein,” as used herein, has the meaning commonly given it inthe art and includes, for example both structural and functional (i.e.,enzymes) proteins. “Protein” includes both natural and syntheticproteins produced or isolated by any means known in the art. Non-naturalproteins are also encompassed by this term. Thus, for example, a proteinmay contain one or more mutations in the amino acid sequence of itspeptide backbone. Proteins may also bear unnatural groups added asprobes or to modify protein characteristics. These groups may be addedby chemical or microbial modification of the protein or one of itssubunits. Additional variations on the term “protein” will be apparentto those of skill in the art.

The term “oligopeptide,” as used herein, refers to a peptide which ismade up of 2-10 amino acid units. “Polypeptide,” as used herein, refersto peptides containing greater than 10 amino acid subunits. Both“oligopeptide” and “polypeptide” refer to both natural and syntheticpeptides which can contain only natural amino acids, only unnaturalamino acids, or a combination of natural and unnatural amino acids.

As used herein, the term “oligonucleotide” refers to synthetic ornatural nucleotide constructs made up of 2-20 nucleic acids. Theoligonucleotide may be composed of either ribonucleic acids,deoxyribonucleic acids or combinations thereof “Oligonucleotides” can bemade up of only natural nucleic acids, only unnatural nucleic acids or acombination of natural and unnatural nucleic acids.

As used herein, the terms “ribonucleic acid,” “deoxyribonucleic acid,”“chromosomes” and “genes” have the meaning normally given to them bythose of skill in the art and also include modified analogs which may beproduced by any means known in the art including, but not limited to,chemical synthesis and microbial synthesis.

The term “carbohydrates,” as used herein, refers to both natural andsynthetic saccharides, oligosaccharides, polysaccharides, glycoproteinsand mucopolysaccharides. Any means known in the art to produce orisolate carbohydrates can be used to provide carbohydrates of use inpracticing the instant invention.

As used herein, the term “noble gas” refers to a rare or inert gas whichis a member of the zero group of the periodic table. Noble gasessuitable for use in the methods of the present invention include thosehaving a nuclear spin, i.e., a non-zero nuclear spin. Examples of suchnoble gases include, but are not limited to, ³He, ²¹Ne, ⁸³Kr, ¹²⁹Xe,¹³¹Xe and combinations thereof. In a preferred embodiment, the noble gasemployed is ¹²⁹Xe, ¹³¹Xe or ³He. Although these noble gases aregenerally preferred, other noble gases may be preferred in differentapplications because of their different physical, chemical associationand magnetic resonance properties. Additionally, in some instances, itmay be preferred to use a combination of noble gases, e.g., ¹²⁹Xe and³He.

In another aspect, the present invention provides a method for studyinga property of a noble gas in a tissue. The method comprises: (a)hyperpolarizing a noble gas; (b) dissolving the hyperpolarized noble gasin a physiologically compatible liquid carrier to form a mixture; (c)contacting the tissue with the mixture from (b); and (d) scanning thetissue by nuclear magnetic resonance spectroscopy, magnetic resonanceimaging, or both, whereby the property of the noble gas in the tissue isstudied.

In this aspect of the invention, the tissue studied may be any tissue ofthe organism. The tissue may be studied in situ or removed from theorganism to which it is native. In preferred embodiments of this aspectof the invention, the tissue studied is a tissue of the central orperipheral nervous system. In particularly preferred embodiments, thetissue is a component of the central nervous system such as the brain,spinal cord, blood-brain barrier or cerebrospinal fluid and the studiedproperty of the noble gas in the tissue may be either a functional or astructural property.

As used herein, the term “property” encompasses NMR parameters,functional properties and structural properties. The term “NMRparameter” refers to frequency shift, chemical shift, scalar coupling,dipolar coupling, relaxation time (e.g., T₁, T_(1p), T₂, T₂*, etc.).Both functional and structural properties can be derived from the NMRparameters of the system under observation.

The term “functional property,” as used herein, refers to the propertiesof a noble gas interacting with a tissue and includes properties suchas, but not limited to, the mechanism of exchange of the noble gasbetween the intracellular and extracellular compartments, the exchangerate of the noble gas between the intracellular and extracellularcompartments of a tissue, the residence time of the noble gas in theintracellular or extracellular compartment, the effect of the noble gason the chemistry or metabolism of the cell and the concentration of thenoble gas in the extracellular or intracellular compartment of thetissue.

As used herein, the term “structural property” refers to the propertiesof a noble gas interacting with a tissue and includes properties suchas, but not limited to, the spatial distribution of the noble gas withinthe intracellular or extracellular compartment of a tissue and thelocation and identity of sites which bind the noble gas within theintracellular compartment, extracellular compartment or the membraneseparating the compartments.

In one preferred embodiment, the property studied is the mechanism ofexchange of the noble gas between the intracellular and extracellularcompartments of a tissue. In another preferred embodiment, the tissuestudied is a tissue of the peripheral or central nervous system. In amore preferred embodiment, the property studied is the mechanism ofnoble gas exchange between the intracellular and extracellularcompartments of a tissue of the central nervous system.

In still another aspect, the invention provides a method for enhancingthe relaxation time of a hyperpolarized noble gas in contact with aphysiological fluid. In this aspect, the method of the inventioncomprises: (a) forming a hyperpolarized noble gas intermediate solutionby dissolving the hyperpolarized noble gas in a fluid in which therelaxation time of the hyperpolarized noble gas is longer than therelaxation time of the noble gas in the physiological fluid; and (b)contacting the physiological fluid with the intermediate solution.

As used herein, the term “physiological fluid” encompasses the variousintracellular and extracellular fluids which are found in an organism.Such physiological fluids include, but are not limited to, blood,plasma, lymph, cerebrospinal fluid, bile, saliva, gastric fluids,vitreous humor, cytoplasm, etc.

As used herein, the term “relaxation time” refers to the time requiredfor a nucleus which has undergone a transition into a higher energystate to return to the energy state from which it was initially excited.Regarding bulk phenomena, the term “relaxation time” refers to the timerequired for a sample of nuclei, the Boltzmann distribution of which hasbeen perturbed by the application of energy, to reestablish theBoltzmann distribution. The relaxation times are commonly denoted T₁ andT₂. T₁ is referred to as the longitudinal relaxation time and T₂ isreferred to as the transverse relaxation time. Other relaxation times ofrelevance include, but are not limited to T_(1p) (the paramagneticcontribution to the longitudinal relaxation rate) and T₂* (thetransverse relaxtion time including the effect of B₀ inhomogeneity). Asused herein, the term “relaxation time” refers to the above-describedrelaxation times either together or in the alternative. Other relevantrelaxation times will be apparent to those of skill in the art. Anexhaustive treatise on nuclear relaxation is available in Banci, L, etal. NUCLEAR AND ELECTRON RELATION, VCH, Weinheim, 1991, which is hereinincorporated by reference.

In a preferred embodiment of this aspect of the invention, the fluidinto which the hyperpolarized noble gas is dissolved is a fluorocarbonor lipid, as described above. In a more preferred embodiment, the fluidis an aqueous emulsion of either a fluorocarbon or a lipid, or is anaqueous emulsion of a combination of a fluorocarbon and a lipid.

In an additional aspect the invention provides a method for measuring asignal transferred from a hyperpolarized noble gas atom to a non-noblegas NMR active nucleus, comprising: (a) contacting the non-noble gas NMRactive nucleus with the hyperpolarized noble gas atom; (b) applyingradiofrequency energy to the non-noble gas NMR active nucleus in amagnetic field; and (c) measuring the signal transferred from thehyperpolarized noble gas atom to the non-noble gas NMR active nucleususing nuclear magnetic resonance spectroscopy, magnetic resonanceimaging, or both.

In preferred embodiments, the non-noble gas nucleus is a biologicallyrelevant nucleus such as, but not limited to, ¹H, ¹³C, ¹⁵N, ³¹P, etc. Ina particularly preferred embodiment, the nucleus is a proton.

In yet another aspect, the invention provides a pulse sequence forheteronuclear difference spin polarization induced nuclear Overhausereffect (SPINOE) NMR of a system comprising a hyperpolarized noble gasand a non-noble gas NMR active nucleus. The pulse sequence comprises:(a) a non-noble gas NMR active nucleus π/2 pulse; (b) a non-noble gasNMR active nucleus π pulse applied simultaneously with application of anoble gas π pulse; and (c) a non-noble gas NMR active nucleus π/2 pulse.

As used herein, the term “non-noble gas π pulse” denotes aradiofrequency pulse, at the resonant frequency of a non-noble gasnucleus, which is delivered to the system and is of a durationsufficient to rotate the bulk magnetization of the sample of non-noblegas nuclei by 180°. Similarly, a “noble gas π pulse” refers to aradiofrequency pulse sufficient to rotate the bulk magnetization ofnoble gas sample by 180°. A “non-noble gas NMR active nucleus π/2 pulse”will rotate the bulk magnetization of a sample of protons by 90°. Meansof delivering these pulses to the system under observation will beapparent to those of skill in the art.

The pulse sequence embodied in this aspect of the invention can be usedto obtain information related to the transfer of polarization from ahyperpolarized noble gas to a non-noble gas NMR active nucleus such as aproton. In a preferred embodiment, the pulse sequence is used to studyregions of a structure that bind to or otherwise interact with thehyperpolarized noble gas. In other preferred embodiments, the pulsesequence is used to study a macromolecule such as a protein,polysaccharide, polypeptide, oligonucleotide, or any other moleculewhich interacts with a hyperpolarized noble gas in a NMR or MRIdiscernable manner. In still another preferred embodiment, thehyperpolarized noble gas is dissolved in a fluid prior to itsadministration to a tissue.

In another embodiment, the invention provides an apparatus for preparinga solution of a hyperpolarized noble gas. The apparatus comprises: avessel for receiving the fluid; a reservoir for receiving thehyperpolarized noble gas, the reservoir communicating through a firstshutoff valve with the vessel, the reservoir being shaped to allow thereservoir to be cooled independently of the vessel; a gas inlet portcommunicating through a second shutoff valve with the reservoir; and ameans for withdrawing the fluid from the vessel independently of theshutoff valve.

The apparatus can be constructed of any material with the caveats thatthe material does not speed the relaxation of the hyperpolarized gas andmust be capable of withstanding the temperatures necessary to freeze thenoble gas and the temperature shifts between the temperature used tofreeze the noble gas and room temperature or higher. Thus, the apparatuscan be constructed of, for example, glass, pyrex, metal or plastic.

The limitations on the shape and size of the components are minimal. Theonly essential limitation being that the noble gas reservoir is capableof being cooled separately from the fluid vessel. Thus, it is within thescope of the invention to have a reservoir which is a side-arm, flask orother receptacle pendent off of the fluid vessel. The reservoir may alsobe separable from the rest of the apparatus by means of a joining meanssuch as, for example, tubing, hoses, ground-glass joints,ball-and-socket joints, or any other joining means known to those ofskill in the art. When large volumes of gas and/or fluid are to be used,it is particularly preferred that the apparatus be composed of separablecomponents (i.e., reservoir and vessel) which can be assembled anddisassembled as need be to facilitate the purpose of the apparatus.

The shutoff valves between the main components of the apparatus compriseany means of reversibly separating two attached vessels known in theart. Thus, it is within the scope of the invention to use a stopcock,septum, valve, check-valve, pressure-release valve, etc. Similarly, themeans to remove the fluid from the vessel may comprise any means knownin the art to reversibly seal a vessel. These include, but are notlimited to, stopcocks, septa, membranes, break-seals, caps, plugs,break-seals, etc.

In a preferred embodiment, the apparatus further comprises a means forfreezing the hyperpolarized noble gas in the reservoir for receiving thehyperpolarized noble gas. The means to freeze the gas may consist of anymeans known in the art for attaining temperatures sufficiently low tofreeze a noble gas. These include, but are not limited to, liquid gases,circulating baths and refrigeration units.

In another preferred embodiment, the apparatus further comprises a meansfor applying a magnetic field to the frozen hyperpolarized noble gas topreserve the hyperpolarization prior to forming the mixture between thehyperpolarized gas and the fluid. Any means known in the art forapplying a magnetic field will be useful in the instant invention. Theseinclude, but are not limited to, permanent magnets, electromagnets,superconducting magnets and the magnet in an NMR spectrometer or imagingdevice.

As noted above, the noble gas used in the methods of the presentinvention is hyperpolarized relative to its normal Boltzmannpolarization. Noble gases can be hyperpolarized for use in accordancewith the present invention through any of various means known to andused by those of skill in the art. Such methods: include, but are notlimited to, spin-exchange interactions with optically pumped alkalimetal vapor and direct pumping by a metastable state. It will be readilyapparent to those of skill in the art that other methods can also beused to hyperpolarized the noble gases used in the present invention. Ina preferred embodiment, optical pumping using circularly polarized lightis used to produce a hyperpolarized gas.

The term “optical pumping” generally refers to the redistribution ofatoms among their fine- or hyperfine-structure levels by means of light.The light can be circularly polarized, anisotropic, filtered oramplitude-modulated. In preferred embodiments, the light is circularlypolarized. Using relatively simple techniques known to those of skill inthe art, it is possible to produce useful polarization of atoms, nucleiand electrons. For example, see, Carver, T. R., Science,141(3581):599-608 (1963), for a detailed review of optical pumping Inaddition, the details of an optical-pumping apparatus suitable for usein accordance with the present invention are described, for example, byRaftery, et al., Phys. Chem., 97: 1649 (1993); and Song, et al., J.Magnet. Res. 115: 127-130 (1995). The teachings of the above-citedreferences are incorporated herein by reference.

The optical pumping and spin-exchange can be performed in the absence ofan applied magnetic field, but are preferably performed using modestfields of about 1 G or larger. Pumping in the NMR magnet bore at fieldsof several Tesla is also possible. The maximum steady state nuclearpolarization achievable depends on the time constant characterizing thespin exchange with the alkali metal and the time constant characterizingthe relaxation (T₁) due, for example, to contact with the surfaces ofthe pumping cell. For instance, with ¹²⁹Xe, T₁=20 min. polarizations of20-40% are quite practicable, and polarizations of 90% or more should beattainable.

Hyperpolarizing noble gases through spin exchange with an opticallypumped alkali-metal vapor starts with the irradiation of thealkali-metal vapor with circularly polarized light at the wavelength ofthe first principal (D₁) resonance of the alkali metal (e.g., 795 nm forRb). The ²S_(1/2) ground state atoms are thus excited to the ²P_(1/2)state and subsequently decay back to the ground state. If performed in amodest (10 Gauss) magnetic field aligned along the axis of incident D₁light, this cycling of atoms between the ground and first excited statesleads to nearly 100% polarization of the atoms. This polarization iscarried mostly by the lone valence electron characteristic of all alkalimetals; this essentially means that all of these electrons have theirspin either aligned or anti-aligned to the magnetic field depending uponthe helicity (right- or left-handed circular polarization state) of thepumping light. If a noble gas with non-zero nuclear spin is alsopresent, the alkali-metal atoms can undergo collisions with the noblegas atoms in which the polarization of the valence electrons istransferred to the noble-gas nuclei through a mutual spin flip. Thisspin exchange results from the Fermi-contact hyperfine interactionbetween the electron and the noble-gas nucleus. By maintaining thealkali-metal polarization at nearly 100% with the pumping light, largenon-equilibrium polarizations (5%-80%) are currently achievable in largequantities of a variety of noble gases through this spin-exchangeprocess. For example, one currently available Titanium:Sapphire-lasercan theoretically provide 1 g/hr (200 cc-atm/hr) of highly polarized¹²⁹Xe. Even more product is expected from the use of modem diode arraylasers.

The alkali metals capable of acting as spin exchange partners inoptically pumped systems include any of the alkali metals. Examples ofalkali metals suitable for use in this hyperpolarization techniqueinclude, but are not limited to, ²³Na, ³⁹K, ⁸⁵Rb, ⁸⁷Rb and ¹³³Cs. In apresently preferred embodiment, ⁸⁵Rb and ⁸⁷Rb are the alkali metalisotopes employed.

In addition to optical pumping, the noble gas may be hyperpolarizedusing metastability exchange. The technique of metastability exchangeinvolves direct optical pumping of, for example, ³He, without need foran alkali metal intermediary. The method of metastability exchangeusually involves the excitation of ground state ³He atoms (1¹S₀) to ametastable state (2³S₁) by weak radio frequency discharge. The 2³S₁atoms are then optically pumped using circularly polarized light havinga wavelength of 1.08 μm in the case of ³He. The light drives transitionsup to the 2³P states, producing high polarizations in the metastablestate to which the 2³P atoms then decay. The polarization of the 2³S,states is rapidly transferred to the ground state through metastabilityexchange collisions between metastable and ground state atoms.Metastability exchange optical pumping will work in the same lowmagnetic fields in which spin exchange pumping works. Similarpolarizations are achievable, but generally at lower pressures, e.g.,about 0-10 Torr.

Prior to and independent of hyperpolarization, further enhancement ofthe noble gas magnetic resonance signal can be obtained by increasingthe proportion of the NMR active isotope in each noble gas to a levelabove the natural abundance of such imageable isotopes in the noble gas.For instance, in the case of ¹²⁹Xe, which has a natural isotopicabundance of about 26%, the enhancement can amount a factor of aboutfour for a gas which is enriched to 100% ¹²⁹Xe. Thus, althoughhyperpolarization plays a much larger role in signal enhancement,isotopic enrichment can provide a significant contribution to theultimate efficacy of the present invention.

In the methods of the present invention, the hyperpolarized noble gas,e.g., ¹²⁹Xe, can be delivered in gas, liquid or solid phases. Highpressure noble gas can be conveniently obtained by first freezing into asmall volume in the magnetic field followed by warming up. The noble gasis then combined with a fluid to form a mixture. Such a mixture can beformed, for example, by vigorous shaking to equilibrate quickly thenoble gas in the liquid, or by other efficient means of gas/liquidmixing which are known to and used by those of skill in the art.Alternatively, porous membranes or other devices known to those of skillin the art can be used to saturate the solution with the noble gas,provided they do not significantly decrease the relaxation time of thenoble gas. It should be noted that the freezing of the hyperpolarizednoble gas also serves to purify the noble gas, e.g., to remove orseparate out the toxic alkali metal used in the hyperpolarization, andto prolong the hyperpolarization of the noble gas during storage orshipping.

In the methods of the present invention, magnetic resonance spectroscopyand/or magnetic resonance imaging is used to detect a parameter whichcan be used to analyze, characterize or image a sample or a portionthereof. Parameters of the sample, the hyperpolarized noble gas or thesystem comprising the sample and the hyperpolarized noble gas, which areuseful for such purposes include, but are not limited to, chemicalshift, T₁ relaxation, T₂ relaxation and T_(1ρ) relaxation. In apreferred embodiment, multiple parameters are detected. In addition,multiple techniques can be employed in the methods of the presentinvention to collect and manipulate nuclear magnetic resonance data.Such methods include, but are not limited to, one-dimensional andmulti-dimensional spectroscopy, Fourier imaging, planar imaging,echo-planar imaging (EPI), projection-reconstruction imaging, spin-warpFourier imaging, gradient recalled acquisition in the steady state(GRASS) imaging also known as fast low angle shot (FLASH) imaging, andhybrid imaging. For imaging purposes, preferred methods include theFLASH or GRASS imaging method and the EPI method because of theircapacity to generate images through fast data acquisition, therebyconserving polarization of the noble gas.

The methods of the present invention can be used for a myriad of diverseapplications including, but not limited to, tissue perfusionquantitation; longer residence time imaging of air space; new protoncontrast agent; new probe of pathophysiology; new application of NMR togastrointestinal clinical medicine; new non-toxic intravascular MRIangiography contrast agent; and protein structure elucidation bypolarization transfer to protons or other nuclei in the molecule. Inaddition, the noble gas, when dissolved in a physiologically acceptablecarrier can be utilized to study lung air-space anatomy, tissueperfusion and MRI angiography. Moreover, within the context of themethods disclosed herein, the present invention also has the followingadvantages. In general, hyperpolarized noble gas NMR can be used as analternative to the imaging techniques that make use of radioactiveisotopes, such as ¹²⁷Xe and ¹³³Xe. The advantages of MRI ofhyperpolarized noble gases are the zero radiation dose absorption by thepatient and, in addition, a much better spatial resolution. In addition,NMR of noble gases are useful for brain studies. Specifically, magneticresonance imaging of a hyperpolarized noble gas enables better detectionof central nervous system perfusion and, thus, it is useful as a toolfor diagnosis of stroke and as a flow specific tool for functionalimaging. Those of skill in the art will readily appreciate that themethods of the present invention are useful for a variety of otherpurposes as well.

Those of skill in the art will readily appreciate that the noble gas ispreferably maintained in a system which is substantially sealed toprevent loss to the atmosphere. Typically, a sealed containmentapparatus will include a noble gas source, such as a gas canister orcompressed gas tank, conduits to and away from a sample, as well asrecovery apparatus. Moreover, a hyperpolarized noble gas may be storedfor extended periods of time in a hyperpolarized state. Storage systemscapable of cryogenic storage of a hyperpolarized noble gas arepreferably able to maintain tempera such that noble gas is stored infrozen state. For instance, frozen ¹²⁹Xe can be reasonably maintained atfields of ≧500 Gauss at temperatures ranging from 4.2K (liquid heliumtemperature), for which T₁, is about a million seconds (10 days), to 77K(liquid nitrogen temperature), for which T₁, is about 10 thousandseconds. The fields necessary here may be provided by a permanentmagnet, a larger electromagnet or a superconducting magnet. Those ofskill in the art will readily appreciate that a noble gas which has beenhyperpolarized by spin exchange with an alkali metal may be storedeither before or after removal of any alkali metal used in spin exchangehyperpolarization techniques. In all cases in which rubidium or otheralkali metal would interfere with the behavior of the system, the alkalimetal is removed before introduction of the noble gas to the sampleusing techniques known to and used by those of skill in the art.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are intended neither to limit or define the invention in any manner.

EXAMPLES

Materials and Methods

The following general materials and methods were used in the examplesdescribed below.

The design of the shaker used in the dissolution stage for xenon mixingand delivery is illustrated in FIG. 1. The shaker has a small sidearmwhich can be isolated from the main volume by a stopcock The shaker ischarged with a sample of either normal abundance or isotopicallyenriched xenon (80% ¹²⁹Xe, EG&G Mound, Miamisburg, Ohio, U.S.A.). Laserpolarization is performed prior to admitting the xenon to the shaker.Briefly, approximately 5×10⁻⁴ mol of 80% isotopically enriched ¹²⁹Xe wasoptically pumped in a 30 cc cylindrical glass pumping cell (diameter≈30mm). Before optical pumping, the cell was cleaned and coated withSURFASIL® (Pierce Chemical Co., Florence, Mass., U.S.A.); the cell wasthen evacuated to 10⁻⁶ torr and loaded with one drop of melted rubidiummetal in a dry nitrogen environment. Optical pumping was performed witha 1.3 W continuous-wave Ti:sapphire laser (794.7 nm) for 20-30 min, and

EXAMPLES

Materials and Methods

The following general materials and methods were used in the examplesdescribed below.

The design of the shaker used in the dissolution stage for xenon mixingand delivery is illustrated in FIG. 1. The shaker has a small sidearmwhich can be isolated from the main volume by a stopcock. The shaker ischarged with a sample of either normal abundance or isotopicallyenriched xenon (80% ¹²⁹Xe, EG&G Mound, Miamisburg, Ohio, U.S.A.). Laserpolarization is performed prior to admitting the xenon to the shaker.Briefly, approximately 5×10⁻⁴ mol of 80. % isotopically enriched ¹²⁹Xewas optically pumped in a 30 cc cylindrical glass pumping cell(diameter≈30 mm). Before optical pumping, the cell was cleaned andcoated with SURFASIL® (Pierce Chemical Co., Florence, Mass., U.S.A.);the cell was then evacuated to 10⁻⁶ torr and loaded with one drop ofmelted rubidium metal in a dry nitrogen environment. Optical pumping wasperformed with a 1.3 W continuous-wave Ti:sapphire laser (794.7 nm) for20-30 min, and the temperature of the cell was maintained at 60-80° C.by a temperature-controlled nitrogen gas stream. Typically, theapparatus produces xenon polarization levels in the range of 5-10%.

Following laser polarization, the polarized ¹²⁹Xe is frozen at liquidnitrogen temperatures in the sidearm in a magnetic field ofapproximately 50 mT provided by a small permanent magnet. The magneticfield is used in the freezing stage to prevent the decay of xenonpolarization. The xenon is sublimated and then admitted into thesolution. The small size of the shaker allows for the accumulation ofseveral atmospheres of xenon pressure which aids in increasing the xenonconcentration in the solution. During the dissolution procedure, thevessel is vigorously shaken to help dissolve the xenon gas. Theresulting xenon solution is extracted with a syringe through ahigh-pressure rubber septum. In those examples below wherein a solutionNMR study is performed on an in vitro sample, the xenon is immediatelyinjected into an NMR tube which contains the sample to be studied. Theloss of polarization during the injection procedure was found to beinsignificant.

Example 1

This example describes the ¹²⁹Xe NMR of a sample of hyperpolarized ¹²⁹Xedissolved in aqueous saline. The T₁ of xenon was measured in both H₂Osaline and D₂O/saline.

In an NMR tube open to the atmosphere were combined saline andhyperpolarized ¹²⁹Xe. The saline used had a NaCl concentration of 0.9%by weight. ¹²⁹Xe was dissolved in saline as described in the materialsand methods section above. Xenon has a low solubility in saline, with anOstwald coefficient of only 0.0926 (the standard temperature andpressure volume of xenon dissolved in 1 liter of liquid at 1 atmosphereof gas pressure; 1 atm=101.3 kPa). In H₂O/saline, the T₁ of xenon isquite long (66 s at 9.4 T). The ¹²⁹Xe NMR spectrum of a solution of¹²⁹Xe in D₂O saline is displayed in FIG. 2. In saline made with D₂O, theT₁ of xenon is ≈1000 s. Thus, the shorter T₁ of xenon in H₂O saline isdue to dipolar couplings between the hyperpolarized xenon electrons andthe proton nuclear spins.

Example 1 demonstrated the acquisition and characteristics of ¹²⁹Xespectra of hyperpolarized xenon dissolved in aqueous solution.

Example 2

This example demonstrates the use of xenon NMR to study the partition ofxenon between the intracellular and extracellular compartments in asample of human blood. The NMR of xenon in human blood was measuredusing both hyperpolarized and unpolarized xenon.

2.1 Materials and Methods

A sample of human blood was prepared by allowing fresh blood from avolunteer to settle for a few hours and subsequently decanting off aportion of the plasma. The portion removed accounted for approximately30% of the total volume of the blood sample. Following removal of aportion of the plasma, xenon saturated saline (1 mL) was injected intothe red blood cell (RBC) sample (1 mL) and the ¹²⁹Xe NMR was measured.The NMR spectra were measured on a Bruker AM-400 spectrometer.

2.2 Results

The NMR spectrum of non-polarized xenon was measured in an RBC sample(FIG. 3A). Considerable signal averaging was required in order to obtaina spectrum with an acceptable signal-to-noise ratio. The spectrum wasacquired over 1.5 h and is the product of 520 scans. In marked contrast,a spectrum with an excellent signal-to-noise ratio was obtainedfollowing one scan when laser-polarized ¹²⁹Xe was used (FIG. 3B). Thesignal enhancement obtained through using laser-polarized ¹²⁹Xe, ratherthan non-polarized ¹²⁹Xe, was estimated to be approximately 3 orders ofmagnitude.

The NMR spectra of both the laser-polarized and non-polarized ¹²⁹Xe inthe RBC sample display two peaks; 216 ppm and 192 ppm. The peak at 216ppm arises from ¹²⁹Xe which has diffused into the RBC. The peak at 192ppm arises from the ¹²⁹Xe which remains extracellular and is in thesaline/plasma mixture. The significant difference between the xenonchemical shift in the RBC and that in the saline/plasma is primarily dueto the xenon binding to hemoglobin.

Thus, through the use of laser polarized xenon it is possible to rapidlydistinguish between intracellular and extracellular populations of¹²⁹Xe. Further, the significantly improved signal-to-noise ratioobtained in spectra measured on samples containing laser polarized ¹²⁹XeNMR spectra allows the real time observation of the dynamics of thetransfer of the xenon from the saline/plasma mixture into the RBC.

Example 3

Example 3 illustrates the use of NMR spectroscopy to observe thedynamics of the mixing of laser polarized ¹²⁹Xe between theintracellular and extracellular compartments of a sample consisting ofred blood cells and plasma.

3.1 Materials and Methods

A sample of laser polarized xenon in saline and a RBC sample wereprepared as described in Examples 1 and 2, respectively. By using shortrf pulses of small tipping angle, ¹²⁹Xe NMR spectra were acquired as afunction of time after injection of the xenon/saline mixture into theblood. NMR spectra were measured on a Bruker AM-400 spectrometer.

3.2 Results

The results of this experiment are illustrated in FIG. 4. In FIG. 4, themain figure shows the time dependence of the xenon signal in the RBC andin the saline/plasma, normalized by the total signal. The initial riseof the RBC signal and decrease in the saline/plasma signal indicates thetransfer of xenon from the saline/plasma water mixture to the RBC duringthe mixing. Within the first second, the rise in the RBC signal and thereduction of the saline/plasma signal describe the dynamic process ofxenon entering the RBC from the saline/plasma during mixing. The timedependence of both the RBC and saline/plasma signals during the mixingprocess can be described by an exponential function of the form:

$\begin{matrix}{{f(t)} = {A + {B\left( {\exp\left( {- \frac{t}{T}} \right)} \right)}}} & (2)\end{matrix}$where A and B are constants and the time constant (T) for this functionwas estimated to be about 200 ms.

The signal increase (about 1 sec) is probably due to xenon rich blooddripping from the walls of the sample tube into the detection coil aftervigorous mixing. The xenon transfer from the water to the red bloodcells is evident. The timescale for the process is 170±30 ms. When 1 ccof saline water is mixed with 1 cc of red blood cells, the equilibriumdistribution of the integral of the two peaks is approximately 50%.Remarkably, the two peaks decay with the same rate constant (about 5seconds). Spin-lattice relaxation time of xenon in blood measured withconventional NMR yielded two different decay rates for the 2 peaks. Thisis probably an artifact associated with the settling of the red bloodcells during the 12 or more hours of data acquisition required for theconventional experiments. After separation of the erythrocytes from theplasma, the xenon exchange between the two compartments is veryinefficient, and two different relaxation times are observed. When thered blood cells and the plasma are mixed, the exchange is fast enough toyield the same T₁ for the two peaks. The value for the exchange rate wehave measured is consistent with this model. As the experiments havebeen performed in a sample tube open to air, an additional contributionto the decay of the signal may be due to xenon transfer to the air. Suchmechanism would not play a role when the solution is administeredintravascularly to tissues.

The inset in FIG. 4 displays the time dependence of the integrated xenonsignal from both peaks in the spectra. From the decay starting after 2seconds, the T₁ of the two components was found to be approximately 5.0seconds. The initial rise in the total xenon signal intensity during thefirst second, following the vigorous injection and mixing of thexenon/saline solution, was most likely caused by xenon-containingblood/plasma/saline mixture descending from the walls of the sample tubeinto the region of the detection coil. Because the sample was unlikelyto be intimately mixed and equilibrated at the start of the NMRmeasurements, the data acquired in the above-described example reflectprimarily the xenon mixing process between the RBC and thesaline/plasma.

This example illustrates the feasibility of using the techniques of thepresent invention to study the dynamics of noble gas exchange betweenthe intracellular and extracellular compartments of a tissue.

Example 4

Example 4 describes the determination, using NMR spectroscopy, of theintrinsic xenon exchange rate between the RBC and the saline/plasma.

4.1 Materials and Methods

A sample of laser polarized xenon in saline and a RBC sample wereprepared as described in Examples 1 and 2, respectively. By using shortrf pulses of small tipping angle, ¹²⁹Xe NMR spectra were acquired as afunction of time after injection of the xenon/saline mixture into theblood. NMR spectra were measured on a CMX Infinity spectrometer(Chemamagnetics-Otsuka Electronics, Fort Collins, Colo., U.S.A.) at amagnetic field of 4.3 Tesla.

4.2 Results

The xenon exchange rate between the extracellular and intracellularcompartments of a RBC/saline/plasma sample was measured by selectivelyinverting the xenon saline/plasma NMR line and observing the recovery ofthe two signals. The selective inversion was achieved by anamplitude-modulated Gaussian pulse of 1 ms duration centered at thefrequency of the saline/plasma signal. This pulse also reduced theabsolute signal intensities for the RBC and saline/plasma peaks by about50%. A field gradient pulse of 1 ms was applied after the inversionpulse to dephase any components of the transverse magnetization. Afterthe inversion pulse, xenon spectra were taken at fixed time intervalsusing a small tipping angle (20°). Following the addition of thexenon/saline solution into the RBC sample, a delay of 3 seconds beforethe application of the inversion pulse insured that the xenon/RBC systemwas well mixed and equilibrated. The results of the experiment aredisplayed graphically in FIG. 5A and FIG. 5B.

FIG. 5A shows the initial equilibrium spectrum 13 ms before theapplication of the inversion pulse and three of a series of spectrawhich were measured after the selective inversion pulse. The exchange ofxenon from the RBC to the saline/plasma is shown by the increase inamplitude of the saline/plasma signal and the corresponding reduction inthe amplitude of the RBC signal. The time dependence of the signals,S_(RBC) and S_(p1), can be described by the following equations:

$\begin{matrix}{{S_{RBC} = {{\left( {S_{RBC}^{o} + S_{p1}^{o}} \right)\frac{\tau_{RBC}}{\tau_{RBC} + \tau_{p1}}} + {S_{o}{\exp\left( {- \frac{t}{\tau}} \right)}}}},} & (3) \\{{S_{p1} = {{\left( {S_{RBC}^{o} + S_{p1}^{o}} \right)\frac{\tau_{p1}}{\tau_{RBC} + \tau_{p1}}} - {S_{o}{\exp\left( {- \frac{t}{\tau}} \right)}}}},} & (4) \\{{S_{o} = {{S_{RBC}^{o}\frac{\tau_{p1}}{\tau_{RBC} + \tau_{p1}}} - {S_{p1}^{o}\frac{\tau_{RBC}}{\tau_{RBC} + \tau_{p1}}}}},} & (5)\end{matrix}$where τ_(RBC) and τ_(p1) are residence time constants for xenon in theRBC and saline/plasma, and 1/τ=1/τ_(RBC)+1/τ_(p1). S^(o) _(RBC) andS^(o) _(p1) are the initial intensities for the RBC and theplasma/saline components, respectively, immediately after the inversionpulse. The effect of the spin-lattice relaxation is neglected sinceτ<<T₁, making S^(o) _(RBC)+S^(o) _(p1) a constant during the exchangeprocess.

The time dependence of the difference of the two signals, ΔS=S^(o)_(RBC)−S^(o) _(p), is shown in FIG. 5B. From an exponential fit, it wasdetermined that τ=12.0±1 ms. The reduction of the signals due to thefinite tipping angle was taken into account. Given the constraint onτ_(pl)/τ_(RBC) from the ratio of the signals at equilibrium,τ_(RBC)=20.4±2 ms, τ_(p1)=29.1±2 ms were obtained. The time scale forthe diffusion of xenon (τ_(RBC)=20.4 ms) corresponded to the time forthe diffusion of xenon over a distance of 11 μm (a diffusion constant of10⁻⁵ cm²/s was assumed). This distance is slightly larger than thecharacteristic dimension of the RBC. The xenon τ_(RBC) was found to belonger than that for water molecules, which was determined to be 12±2 msat room temperature, Herbst, M. D., et al., Am. J. Physiol., 256:C1097-C1104 (1989).

The above example demonstrates that data relevant to the dynamics of theinteraction between laser polarized xenon and its environment (e.g., amixture of red blood cells and plasma) are accessible using NMRspectroscopy.

Example 5

This example illustrates the preparation and NMR properties of a vehiclefor xenon delivery which consists of a mixture of xenon and an aqueoussuspension of lipid vesicles. An efficient method is provided fordelivery of optically polarized xenon to the vascular system in order toobserve the xenon-129 NMR signal before the xenon polarization hasdecayed. Specifically, the hyperpolarized gas is pre-dissolved insolutions where the xenon has a long spin-lattice relaxation time and,thereafter, the xenon/solution mixture is administered to the blood.

5.1 Materials and Methods

A solution of hyperpolarized xenon in INTRALIPID® was prepared in thesame manner as described for the saline solution of hyperpolarizedxenon, however, the shaker was charged with INTRALIPID® rather thansaline. INTRALIPID® solutions consist of aqueous suspensions of lipidvesicles of approximately 0.1 μm in diameter, which are well toleratedin vivo and are used clinically as nutrient supplements. Commerciallyavailable 20% INTRALIPID® solution (Pharmacia, Uppsala, Sweden) isapproved by the U.S. Food and Drug Administration for use in humans.Importantly, xenon has an approximately 4-fold greater solubility inINTRALIPID® than in saline. The INTRALIPID® solution was charged withlaser polarized ¹²⁹Xe and an aliquot (1 mL) of this solution was addedto human blood (1 mL). The spectra were obtained on a Bruker AM-400spectrometer. The 128×64 image was obtained by the Echo Planar Imagingmethod on a Quest 4300 (Nalorac Cryogenics, Martinez, Calif., U.S.A.)spectrometer.

5.2 Results

The xenon T₁ in the INTRALIPID® solution was measured to be 40±3 s. Thespectrum of the laser polarized xenon, delivered to blood as anIntralipid solution, is shown in FIG. 6A. The predominant feature of thespectrum is a peak at 194 ppm, which corresponds to the xenon in thepure Intralipid solution. Only a small signal is observed at 216 ppm;the signal corresponding to xenon in the RBC (i.e., intracellular). Theratio of the peak corresponding to xenon in the INTRALIPID® solution andthe peak from the intracellular xenon is approximately 6:1. This resultis consistent with a higher affinity of the xenon for the lipids and acorrespondingly inefficient transfer into the RBC. The T₁ decay time ofthe signal at 194 ppm was measured to be 16 s, a factor approximately3-fold larger than the corresponding decay time for xenon in the salinewater/blood mixture. The ¹²⁹Xe signal was so strong in this sample as toallow the direct imaging of the xenon distribution in the mixture. Theacquired image is displayed in FIG. 6A (inset).

Xenon in blood can be utilized to study lung air-space anatomy, tissueperfusion and NMR angiography. In general, hyperpolarized xenon NMRwould be an alternative to the imaging techniques that make use ofradioactive isotopes of xenon, such as ¹²⁷Xe and ¹³³Xe. The advantagesof MRI of hyperpolarized xenon are the zero ionizing radiation doseabsorption by the patient and a potentially much better spatialresolution. NMR of xenon may also prove useful for brain studies.Specifically, magnetic resonance imaging of hyperpolarized xenon wouldenable better detection of central nervous system perfusion and thus bea tool for diagnosis of stroke and also a flow specific tool forfunctional imaging.

This example demonstrates the preparation and the properties ofsolutions of hyperpolarized xenon in lipids. Also demonstrated is theprinciple that lipid solutions of laser polarized xenon can be used todeliver polarized xenon through the blood. The presence of the lipid inthe delivery vehicle both retards penetration of the xenon through theRBC membrane and protects the xenon polarization from rapidly decaying.

The use of different solutions for administering hyperpolarized xenon toblood and tissues is very promising for ¹²⁹Xe Spectroscopic Imaging,Chemical Shift Imaging or in vivo Localized NMR Spectroscopy in tissues.¹²⁹Xe NMR parameters, such as the relaxation times, may prove useful toprobe the state of health of tissues or the malignancy of tumors.Moreover, xenon dissolves readily in fat, and hyperpolarized xenon MRImay be an alternative to conventional proton MRI of fatty tissues.

Example 6

Example 6 demonstrates the utility of perfluorocarbons as deliveryvehicles for laser polarized xenon.

Perfluorocarbon compounds are generally chemically inert and non-toxic.Interestingly, perfluorocarbon emulsions are able to absorb andtransport oxygen and carbon dioxide. A representative perfluorocarbonemulsion, FLUOSOL® (Green Cross, Osaka, Japan), was chosen as apromising prototypical delivery vehicle for xenon. FLUOSOL® is anemulsion which contains 20% perfluorocarbon and is approved by the U.S.F.D.A. for intravascular administration in humans as a blood substitute.

A solution of hyperpolarized xenon in FLUOSOL® was prepared in the samemanner as described for the saline solution of hyperpolarized xenon,however, the shaker was charged with FLUOSOL® rather than saline. TheFLUOSOL® solution was charged with laser polarized ¹²⁹Xe and an aliquot(1 mL) of this solution was added to human blood (1 mL). The spectrawere obtained on a Bruker AM-400 spectrometer.

6.2 Results

FIG. 6B shows a ¹²⁹Xe NMR spectrum acquired after mixing theFLUOSOL®/xenon solution with blood. The peak at 216 ppm corresponds toxenon in the RBC, whereas the broad peak centered around 110 ppm (FIG.6B, inset) arises from xenon in the FLUOSOL® solution. Xenon in pureFLUOSOL® has a chemical shift of 110 ppm and the peak exhibits abroadening which is similar to that observed in the spectrum of thexenon/blood/FLUOSOL® solution. The ratio of the integrated intensitiesof the broad and narrow peaks is approximately 3. The T₁ of the narrowpeak was measured to be 13±1 s. This T₁ is, similar to that measured forxenon in INTRALIPID®; longer than that measured for xenon in theRBC/plasma sample. The results with FLUOSOL® suggest that xenonexchanges between the interior of the RBC and an environmentcharacterized by a xenon relaxation time which is longer than thatexhibited by intracellular xenon. Presumably, the xenon which has thelonger relaxation time resides in the FLUOSOL®. These results haveimplications for the selective MRI/NMR of xenon which has beentransferred to tissues.

Also acquired was a two-dimensional MR image of ¹²⁹Xe dissolved in freshhuman blood (FIG. 7). The image was acquired immediately after the bloodwas mixed with a saline solution saturated with hyperpolarized ¹²⁹Xe.

The above example illustrates that perfluorocarbon emulsions are usefuldelivery vehicles for hyperpolarized noble gases. Also demonstrated isthe feasibility of acquiring an MR image of ¹²⁹Xe dissolved in bloodwhen the ¹²⁹Xe is administered to the blood as a saline solution and,therefore, has a shorter T, than is observed for 129Xe in a fluorocarbondelivery agent.

Example 7

7.1 Materials and Methods

Solutions of hyperpolarized ¹²⁹Xe in partially deuterated benzene (25%C₆D₅H, 75% C₆D₆) were prepared as described above for saline solutionsof ¹²⁹Xe with the exception that the shaker was charged with the benzenesolution rather than saline. Typically, 4×10⁻⁴ mol of enriched ¹²⁹Xe(80%, EG&G Mound) were used in one experiment at a pressure of 1 atm.¹²⁹Xe NMR was performed at 51 MHz on a Quest 4300 spectrometer (NaloracCryogenics, Martinez, Calif., U.S.A.) with a home built probe and atipping angle of 3°. ¹H NMR was performed at 185 MHz with a home builtprobe and a tipping angle of 3°.

Time-resolved two-dimensional MR images of ¹²⁹Xe were obtained using thefast low-angle shot (FLASH) imaging method on the Quest 4300 instrumentusing a tipping angle of 3° for each of the 64 signal acquisitions. Thefrequency-encoding gradient was 3.5 G/mm. The step size of thephase-encoding gradient pulses, which were 500 μs long, was 0.063 G/mm.The diameter of the sample tube was 7 mm and the solution occupied aregion within the tube of length 15 mm. The images were 64×128 pixelimages.

The time-resolved distribution (in seconds) of an unshaken sample ofpartially deuterated benzene was obtained from MRI projections along thetube axis (z). The imaging field gradient for the acquisition of theseimages was 2.6 G/mm.

Two-dimensional MR images of the SPINOE-enhanced ¹H signals wereobtained at 2 and 6 minutes after hyperpolarized ¹²⁹Xe was admitted tothe sample tube containing normal benzene. The images were taken by theecho planar imaging method in 24 ms. The frequency encoding gradient was3.15 G/mm; the phase-encoding gradient pulses were 0.14 G/mm and 50 μslong. The image dimension was 128×32 pixels, and the image waszero-filled to 256×256 pixels in data processing.

The methods used and the results obtained in this example are discussedin detail in Navon, G., et al., Science, 271: 1848-1851 (1996), which isherein incorporated by reference.

7.2 Results

In the following example, the preliminary experiments designed to probethe SPINOE between hyperpolarized xenon and protons in solution aredescribed. When hyperpolarized ¹²⁹Xe is dissolved in liquids, atime-dependent departure of the proton spin from its thermal equilibriumwas observed. The variation in magnetization was an unexpectedmanifestation of the nuclear Overhauser effect (NOE), a consequence ofcross-relaxation between the spins of solution protons and ¹²⁹Xe. SPINOEhas been used to monitor time dependent magnetic resonance images andhigh resolution NMR spectra of solution spins as they encounter themigrating xenon atoms.

The time dependence of the ¹²⁹Xe NMR signal intensity observed whenhyperpolarized ¹²⁹Xe is dissolved in liquid benzene is shown in FIG. 8.The observed spin-lattice relaxation time of ¹²⁹Xe in solution, acombination of the gas and solution relaxation times, is ˜200 s innormal benzene and ˜1000 s in the partially deuterated sample (Moschos,A, et al., J. Magn. Reson. 95: 603 (1991); and Diehl, P., et al., J.Magn. Reson. 88: 660 (1990). The difference between these two valuesdemonstrates the effect of magnetic dipolar coupling between ¹H and¹²⁹Xe spins on the relaxation of the ¹²⁹Xe magnetization; the samecoupling underlies the cross relaxation between the xenon and protonspin systems. For the initial NOE experiments, the partially deuteratedliquids were used to promote the effects of cross relaxation over thepotentially limiting auto-relaxation of the proton spins.

The effect of the dissolved hyperpolarized ¹²⁹Xe on the ¹H magnetizationin liquid benzene is illustrated in FIG. 9. The proton NMR signalexhibits a positive or negative time-dependent NOE, depending on thesign of the ¹²⁹Xe magnetization, which is determined by the helicity ofthe laser light or the orientation of the magnetic field in the opticalpumping stage. The fractional enhancement of the proton magnetizationover its thermal equilibrium value is typically ˜0.1 for benzene, andbetween 0.5 and 2 for the partially deuterated sample.

Based on the theory of the nuclear Overhauser effect, the followingexpression can be derived for the maximum change in the polarization ofthe solvent nuclei (I) due to cross relaxation with the dissolved gas(S):

$\begin{matrix}{\frac{{I_{z}\left( t_{0} \right)} - I_{0}}{I_{0}} = {{- \frac{\sigma_{IS}}{\rho_{I}}}\frac{\gamma_{S}{S\left( {S + 1} \right)}}{\gamma_{I}{I\left( {I + 1} \right)}}\frac{\left\lbrack {{S_{z}\left( t_{0} \right)} - S_{0}} \right\rbrack}{S_{0}}}} & (6)\end{matrix}$where γ_(S) and γ_(I) are the magnetogyric ratios of the nuclear spins,σ_(IS) the cross-relaxation rate, and ρ_(I) is the auto-relaxation rateof the I spins. The cross-relaxation rate σ_(IS) has the same value,σ_(IS)=1.9×10⁻⁶ s⁻¹, for both benzene and partially deuterated benzenesolutions, so the difference in the maximum enhancement of the protonpolarization in these two solutions originates from the different protonrelaxation rates, ρ₁=(20 s)⁻¹ in benzene and ρ_(I)=(160 s)⁻¹ in thepartially deuterated solution. Given the spin quantum numbers and themagnetogyric ratios of the two nuclei, I=S=½, γ₁=2.67×10⁸ rad T⁻¹ s⁻¹,and γ_(S)=−7.44×10⁷ rad T⁻¹ s⁻¹, and the enhancement of the ¹²⁹Xepolarization at the time t_(o) when the proton magnetization reaches itsmaximum (minimum), S_(Z)(t_(o))/S_(o)≈6000, the maximum protonenhancement is estimated to be 0.06 in C₆H₆ and 0.5 in the partiallydeuterated solution, in general agreement with the measured values.

The high spin-polarization and the slow relaxation of ¹²⁹Xe in thesolvent allow for a detailed observation of the dissolution process andthe flow of xenon in the solvent by means of MRI. FIG. 10 showstwo-dimensional MRI projections along the vertical axis of the sampletube. Xenon is found to accumulate first at the bottom of the tube,establishing a gradient in xenon concentration and continues to dissolveinto the benzene as the solution gradually becomes saturated. A detailof this process is shown in FIG. 11, where a series of theone-dimensional image intensities along the tube axis reflects thetime-dependent spatial distribution of the xenon. The descent of xenonin the sample tube occurs because of density differences between thesolution and pure benzene. The heavier xenon-rich regions of thesolution, which form at the top of the solution by diffusion of thexenon into the solvent, gravitate to the lower part of the tube bynatural convection, ultimately filling the tube with saturated xenonsolution.

Because of the SPINOE enhancement of the proton spins proximate to thedissolved hyperpolarized xenon, the xenon concentration gradient isexpected to induce a gradient in the proton magnetization. Indeed, asshown in FIG. 12, the benzene proton magnetization images display a timedependent gradient consistent with the spatial distribution of xenonshown in FIGS. 10 and 11. In fact, differential SPINOE enhancements ofproton NMR can be observed in solutions containing more than onecomponent or in molecules possessing nuclei with different chemicalshifts, making it possible to explore the partitioning and selectiveassociation of the hyperpolarized gas.

The foregoing results indicate that it is possible to image not only thehyperpolarized xenon, but also the environment in which it isaccommodated, a finding which has implications for both materials andmedical applications, for xenon as well as for helium. Because theequilibrium polarization of the solution spins, S_(o) is proportional tothe magnetic field, B_(o), the relative SPINOE is inversely proportionalto B_(o) and is thus expected to be more pronounced at the lowermagnetic fields normally used in medical imaging. Furthermore, since thenuclear Overhauser effect depends on the proximity of the xenon nucleusand the neighboring spins, as well as their relative translationalmotion, a large SPINOE is anticipated in systems where the noble gasatoms are partially immobilized in materials, Miller, J. B., et al.,Macromolecules 26: 5602 (1993), or temporarily bound to molecules suchas proteins, Tilton, R. F., et al., Biochemistry 21, 6850 (1982), evenin the presence of relatively fast proton relaxation. The window is thusopened to other potential applications where xenon may be adsorbed inmaterials, on surfaces, or in biological molecules and organisms.

Example 8

This example illustrates the utility of ¹²⁹Xe—¹H SPINOE spectroscopy forstudying the dynamical and structural characteristics of molecules insolution. That the coupling between laser-polarized ¹²⁹Xe and protons ina p-nitrotoluene solution is due to nuclear spin dipolar couplingmodulated by diffusive motion is demonstrated.

8.1 Materials and Methods

The samples were generally prepared as described in the examples above.The pulse sequence used to obtain SPINOE data is a heteronuclear versionof the difference NOE pulse sequence originally suggested by Stonehound,J., et. al. J. Am. Chem. Soc. 116: 6037 (1994) for homonuclear NOEstudies. One method for observing SPINOEs is simply to acquire theproton signal as a function of time after laser-polarized ¹²⁹Xe isintroduced to the solution. The deviation of the proton signal from itsthermal equilibrium value determines the signal due to SPINOE fromlaser-polarized¹²⁹Xe. However, this method relies on a subtraction oftwo large signals (with and without SPINOE), and this subtraction limitsthe sensitivity of the experiment to only those SPINOE signals greaterthan about one percent of the equilibrium signal. This new sequence isadvantageous compared to the conventional SPINOE method since theequilibrium signal can be suppressed by two orders of magnitude or more.This type of sequence has enabled measurements of NOEs less than 10⁻⁴ ofthe equilibrium signal.

The difference SPINOE sequence is shown in FIG. 13. The saturation ofproton resonances is first achieved by applying a train of proton π/2pulses, and this saturation is maintained with the proton π pulsesduring the mixing time when the SPINOE occurs. The timing of the πpulses is adjusted to give optimal saturation. A π pulse is also appliedto the ¹²⁹Xe resonance at the same time of the proton π pulse so thatthe proton signal due to SPINOE will be accumulated over the entiremixing time. Odd numbers of such π pulse pairs were used so that eachacquisition inverted the ¹²⁹Xe magnetization; thus the subtraction oftwo consecutive signals effectively removed all contributions to thesignal that did not originate from SPINOE.

8.2 Results

Polarization transfer from laser-polarized xenon to p-nitrotoluene in asolution of perdeuterated benzene was observed. p-nitrotoluene is asimple molecule that does not show binding of xenon in solution; thus weanticipated that its couplings of its protons to xenon would be similarto that of benzene protons. The difference SPINOE proton spectra withlaser-polarized ¹²⁹Xe are shown in FIG. 14A and FIG. 14B. The ¹²⁹Xepolarization is negative in FIG. 14A and positive in FIG. 14B and themagnetization transfer to proton is found to be negative and positive,respectively. This observation is consistent with a correlation timethat is much shorter than the inverse of the Larmor frequencies of ¹Hand ¹²⁹Xe, in which case the cross-relaxation constant σ_(IS) would bepositive. From the initial rise of the proton SPINOE signal intensity,we obtain the values of σ_(IS) for the aromatic and methyl protons to besimilar to that for benzene protons and the theoretical estimate of thecross-relaxation rate due to dipolar coupling modulated by moleculardiffusion.

The above example demonstrates the utility of ¹²⁹Xe—¹H SPINOEspectroscopy for studying the dynamical and structural characteristicsof molecules in solution. That the coupling between laser-polarized¹²⁹Xe and protons in a p-nitrotoluene solution is due to nuclear spindipolar coupling modulated by diffusive motion is demonstrated. Further,it has been shown that the sign of the SPINOE signal is influenced bythe sign of the ¹²⁹Xe polarization.

Example 9

This example demonstrates the effect of ¹²⁹Xe binding to a molecule insolution on the observed SPINOE signal(s) arising from that molecule. Acyclic polysaccharide, cyclodextrin, was chosen as a model compound.

9.1 Materials and Methods

Hyperpolarized xenon and mixtures of hyperpolarized xenon andcyclodextrins were prepared generally as discussed above. SPINOE signalsof 0.05 M cyclodextrin solutions in deuterated DMSO were measured asdescribed above in Example 8.

9.2 Results

α-Cyclodextrin is a naturally occurring host molecule composed of sixD-glucose units linked head to tail in a 1α, 4-relationship to form aring known as a cyclohexaamylose. It has a relatively inflexibledoughnut shaped structure where the top of the molecule has twelvehydroxyl groups from positions 2 and 3 of the glucose units and thebottom has the 6 primary hydroxyl groups from position 6. Theequilibrium proton spectrum of α-cyclodextrin in deuterated DMSO isdisplayed in FIG. 15. Cyclodextrins are cyclic glucopyranose oligomersthat possess a hydrophobic binding pocket, Saenger, W., Angew. Chem.Int. Ed. 19: 344 (1980). The hydrophobic binding properties ofcyclodextrins permit them to complex a number of different guestspecies, from drugs to noble gases, Szejtli, J., CYCLODEXTRINTECHNOLOGY, Kluwer-Academic, Dordrecht, 1988. Specifically, it has beenshown in NMR studies that α-cyclodextrin complexes xenon, Bartik, K., etal., J. Magn. Res. B, 109: 164 (1995).

The first evidence of strong couplings between xenon and α-cyclodextrinis the reduced ¹²⁹Xe T₁ in the solution of α-cyclodextrin. For example,the measured ¹²⁹Xe T₁ was 20 s in 0.1 M α-cyclodextrin solution indeuterated DMSO, compared to a T₁>500 s in 0.1 M p-nitrotoluene indeuterated benzene. This increase in the apparent relaxation rate ofxenon is due to the dipolar coupling between xenon and the protons ofα-cyclodextrin; this coupling not only determines the cross-relaxationof the two spins, but also contributes to the xenon auto-relaxation.

In order to study the effects of xenon binding on ¹²⁹Xe—¹H SPINOE,SPINOEs from laser-polarized xenon to α-cyclodextrin dissolved in asolution of perdeuterated dimethyl sulfoxide (DMSO) were observed. Theproton SPINOE spectra of α-cyclodextrin in the presence of ¹²⁹Xe ofnegative polarization and of positive polarization are shown in FIG. 16and FIG. 17, respectively. The assignment of the proton resonance hasbeen reported in other work, Djedaini, F., et al., J. Mol. Struct., 239:161 (1990). In contrast to the SPINOE spectra of p-nitrotoluene, theSPINOE signal intensities for various protons of α-cyclodextrin aresubstantially different. The strongest SPINOEs are observed from H3 andH5, protons located on the inside of the cyclodextrin cavity. The SPINOEsignals from the outer protons H2, H4, and H1, however, are about afactor of 6 smaller. This difference in the xenon coupling to variousprotons can be expected because such dipolar coupling is highlysensitive to the relative distance between spins. One can derive a ratioof the distances between xenon-H3,H5 and xenon-HI,H2,H4 to be1/⁶√{square root over (6)}=1/1.35.

The percentage SPINOE signal is significantly larger than that fromp-nitrotoluene solution. Taking into account the xenon pressures in thesample cell and the magnetic fields applied in the different experimentsof p-nitrotoluene and α-cyclodextrin, we estimate that the ratio of thecross-relaxation rates of α-cyclodextrin and p-nitrotoluene isapproximately 100. This large increase in the overall coupling constantcan be attributed to significant binding between xenon andα-cyclodextrin molecules. Although smaller, the SPINOE signals from thethree hydroxyl protons are also observable.

Additionally, the xenon coupling constants have been compared forα-cyclodextrin and β-cyclodextrin, where β-cyclodextrin is a seven-unitcyclodextrin ring. Even though the size of β-cyclodextrin is merely 15%larger than α-cyclodextrin, its binding of xenon is dramatically reducedand the coupling constants are two orders of magnitude smaller,essentially equivalent to the coupling constants of p-nitrotoluene.

In the above example, it was demonstrated that the off-equilibriumpolarization of xenon can be transferred to other nuclear species, suchas protons. Thus, hyperpolarized xenon can be exploited as a contrastagent for protons. Moreover, it can be used to elucidate structures ofbiologically relevant molecules, such as proteins, by selectivepolarization transfer to the protons of the specific sites where thexenon binds.

Example 10

This example describes the in vivo use of hyperpolarized xenon dissolvedinto a lipid vehicle. Optically pumped xenon was dissolved into a lipidemulsion as described in Example 5 and injected intravenously into arat. The ¹²⁹Xe NMR spectra from the region of the heart and liver wererecorded as a function of time.

10.1 Materials and Methods

The laser polarized xenon and the solution of laser polarized xenon inINTRALIPID® were prepared essentially as described in the precedingexamples.

Male rats weighing 200-250 grams were anesthetized by intramuscularinjection of ketamine/xylazine/acepromazine (30/3/0.6 mg/kg).Supplemental intramuscular doses were administered as needed to maintainanesthesia. A venous catheter was placed into a tail vein, and thereceiver/transmitter surface coil was placed over the heart and liver(FIG. 18). Acquisitions began at start of the injection. Prior to eachexperiment the rat was placed in lateral recumbency into the magnet. Atthe conclusion of each experiment, the catheter was removed and the ratwas returned to its cage to recover from anesthesia.

The ¹²⁹Xe NMR spectra were obtained on a home-built NMR spectrometerinterfaced with a Bruker 2.35 T magnet (xenon frequency: 27.68 MHz, borediameter: 25 cm). The receiver-transmitter surface coil had a diameterof 3.5 cm. For the spectroscopy experiment, spectra were obtained everysecond (pulse angle: ≈20°).

10.2 Results

A series of xenon NMR spectra were taken from the beginning of theintravenous injection of the xenon/INTRALIPID® solution. A spectrumrepresenting an average of the sixth through twelfth scans is shown inFIG. 19; the time-dependence of the integrated signal is shown in theinset. It was anticipated that the Intralipid would initially accumulatein the liver; it is likely that the initial rise in signal amplitudereflects this accumulation, while the subsequent decay is due towash-out, xenon relaxation, and the application of rf pulses.

This example demonstrates the feasibility of using lipid solutions ofhyperpolarized xenon to deliver the xenon via an intravenousadministration route. Also illustrated is that in vivo spectra of thehyperpolarized xenon can be readily obtained.

Example 11

This example describes the use of ¹²⁹Xe MR imaging to obtain images ofthe in vivo distribution of hyperpolarized xenon in the rat. Thehyperpolarized xenon was administered intramuscularly as a salinesolution.

11.1 Materials and Methods

The methods for preparing the hyperpolarized xenon and a saline solutionof the hyperpolarized xenon have been described in previous examples.The rats, anesthesia and apparatus were as described in Example 10,above. For the imaging experiment, the catheter was placed in the muscleof the rats thigh and secured with tape. The surface coil was placedover the injection site on the rat's thigh. At the conclusion of theexperiment, the catheter was removed and the rat was returned to thecage to recover from anesthesia.

Axial images were acquired perpendicular to the coil using the FLASHsequence shown in FIG. 20. In the imaging experiment, tentwo-dimensional ¹²⁹Xe MR images were taken at intervals of approximately7 s (with the exception of an 18 s delay between images 5 and 6) fromthe beginning of the injection of the xenon/saline solution.

11.2 Results

Six of the images obtained are shown in FIG. 21, and depict the signalintensity of the optically pumped xenon in the upper part of the rat'shind leg. The central region of low xenon signal intensity likelycorresponds to the rat's femur. From the six images, one may observethat the signal intensity rises quickly and reaches a maximum at thesecond image (b) (7 s after the start of the injection), and then decaysin the following images. The initial rise in intensity is due to theaccumulation of the xenon/saline solution from the injection, while thesubsequent decay is due mostly to the application of the rf pulses (48pulses of approximately 5 degrees tipping angle), although xenonrelaxation and wash-out undoubtedly made additional contributions tothis decay. The change in the pattern of the images suggests that partof the xenon/saline solution may have penetrated and diffused into thesurrounding tissue over the duration of the experiment.

The major advantage of saline water as the xenon solvent is the longxenon T₁, which permits negligible loss of polarization over theinjection time. However, the solubility of xenon in saline water is lowwith an Ostwald coefficient of only 0.0926 (the STP volume of xenondissolved in 1 liter of liquid at 1 atm of gas pressure). Higher xenonconcentrations can be obtained by using alternative xenon solvents (e.g.INTRALIPID® and FLUOSOL®). Furthermore, the xenon partitioningproperties of such solvents in biological tissues allow particular invivo applications. It was determined in previous in vitro studies thatsuch solvents can bring about a three-fold increase in the effectiverelaxation time of xenon in blood. Thus, administration of the polarizedxenon dissolved into one of these two classes of delivery vehicles isanticipated to improve the MR images acquired and to afford a longertemporal imaging window.

Example 11 demonstrates that in vivo ¹²⁹Xe MR images can be obtained andused to study the distribution of hyperpolarized xenon in a livingsystem.

It is to be understood that the above description and examples areintended to be illustrative and not restrictive. Many embodiments willbe apparent to those of skill in the art upon reading the abovedescription and examples. The scope of the invention should, therefore,be determined not with reference to the above description and examples,but should instead be determined with reference to the appended claims,along with the full scope of equivalents to which such claims areentitled. The disclosures of all articles and references, includingpatent applications and publications, are incorporated herein byreference for all purpose.

1. An apparatus for preparing a solution of a hyperpolarized noble gasin a fluid, comprising: a vessel for receiving the fluid; a reservoirfor receiving the hyperpolarized noble gas, the reservoir communicatingthrough a first shutoff valve with said vessel, said reservoir beingseparated in space from said vessel such that it can be cooledindependently of said vessel; a gas inlet port communicating through asecond shutoff valve with said reservoir; means for withdrawing saidfluid from said vessel independently of said first shutoff valve andsaid second shutoff valve; a cryogenic bath of liquid gases for freezingsaid hyperpolarized noble gas; and a magnet selected from the group ofmagnets consisting essentially of a permanent magnet, an electromagnet,a superconducting electromagnet, and a shimmed NMR spectrometer forapplying a magnetic field to said reservoir.
 2. An apparatus as recitedin claim 1, wherein said means for withdrawing said fluid from saidvessel comprises: a reversible seal coupled to a port in said vessel;and a syringe configured to be removably inserted through saidreversible seal.
 3. An apparatus as recited in claim 2, wherein saidreversible seal comprises a seal selected from the group of sealsconsisting essentially of a cap, a plug, a septa, a membrane, and abreak seal.
 4. An apparatus as recited in claim 1, wherein said meansfor withdrawing said fluid from said vessel comprises: a fluid flowregulator coupled to a port in said vessel; and a tubular memberassociated with said fluid flow regulator.
 5. An apparatus as recited inclaim 4, wherein said fluid flow regulator comprises a valve selectedfrom the group of valves consisting essentially of a stopcock, a checkvalve, a ball valve, and a pressure release valve.
 6. An apparatus forpreparing a solution of a hyperpolarized noble gas in a fluid,comprising: a vessel for receiving the fluid; a reservoir for receivingthe hyperpolarized noble gas, the reservoir communicating through afirst shutoff valve with said vessel, said reservoir being separated inspace from said vessel such that it can be cooled independently of saidvessel; a gas inlet port communicating through a second shutoff valvewith said reservoir; means for withdrawing said fluid from said vesselindependently of said first shutoff valve and said second shutoff valve,wherein said means for withdrawing comprises a reversible seal coupledto a port in said vessel; means for freezing said hyperpolarized noblegas; means for applying a magnetic field to said reservoir.
 7. Anapparatus as recited in claim 6, wherein said means for withdrawing saidfluid from said vessel comprises: a syringe configured to be removablyinserted through said reversible seal.
 8. An apparatus as recited inclaim 6, wherein said reversible seal comprises a seal selected from thegroup of seals consisting essentially of a cap, a plug, a septa, amembrane, and a break seal.
 9. An apparatus as recited in claim 6,wherein said means for freezing said hyperpolarized noble gas comprisesa cryogenic bath of liquid gases.
 10. An apparatus as recited in claim6, wherein said means for freezing said hyperpolarized noble gascomprises a refrigeration unit.
 11. An apparatus as recited in claim 6,wherein said means for applying a magnetic field to said reservoircomprises a magnet selected from the group of magnets consistingessentially of a permanent magnet, an electromagnet, a superconductingelectromagnet, and a shimmed NMR spectrometer.
 12. An apparatus forpreparing a solution of a hyperpolarized noble gas in a fluid,comprising: a vessel for receiving the fluid; a reservoir for receivingthe hyperpolarized noble gas, the reservoir communicating through afirst shutoff valve with said vessel, said reservoir being separated inspace from said vessel such that it can be cooled independently of saidvessel; a gas inlet port communicating through a second shutoff valvewith said reservoir; means for withdrawing said fluid from said vesselindependently of said first shutoff valve and said second shutoff valve,wherein said means for withdrawing comprises a fluid flow regulatorcoupled a port in said vessel; means for freezing said hyperpolarizednoble gas; and means for applying a magnetic field to said reservoir.13. An apparatus as recited in claim 12, wherein said means forwithdrawing said fluid from said vessel comprises: a tubular memberassociated with said fluid flow regulator.
 14. An apparatus as recitedin claim 13, wherein said fluid flow regulator comprises a valveselected from the group of valves consisting essentially of a stopcock,a check valve, a ball valve, and a pressure release valve.
 15. Anapparatus as recited in claim 12, wherein said means for applying amagnetic field to said reservoir comprises a magnet selected from thegroup of magnets consisting essentially of a permanent magnet, anelectromagnet, a superconducting electromagnet, and a shimmed NMRspectrometer.
 16. An apparatus as recited in claim 12, wherein saidmeans for freezing said hyperpolarized noble gas comprises a cryogenicbath of liquid gases.
 17. An apparatus as recited in claim 12, whereinsaid means for freezing said hyperpolarized noble gas comprises arefrigeration unit.
 18. An apparatus for preparing a solution of ahyperpolarized noble gas in a fluid, said apparatus comprising: a vesselfor receiving the fluid; a reservoir for receiving the hyperpolarizednoble gas, the reservoir communicating through a first shutoff valvewith said vessel, said reservoir being separated in space from saidvessel such that it can be cooled independently of said vessel; a gasinlet port communicating through a second shutoff valve with saidreservoir; means for withdrawing said fluid from said vesselindependently of said first shutoff valve and said second shutoff valve;means for freezing said hyperpolarized noble gas; and means for applyinga magnetic field to said reservoir.
 19. An apparatus as recited in claim18, wherein said means for withdrawing said fluid from said vesselcomprises: a reversible seal coupled to a port in said vessel; and asyringe configured to be removably inserted through said reversibleseal.
 20. An apparatus as recited in claim 19, wherein said reversibleseal is a seal selected from the group of seals consisting essentiallyof a cap, a plug, a septa, a membrane, and a break seal.
 21. Anapparatus as recited in claim 19, wherein said means for withdrawingsaid fluid from said vessel comprises: a fluid flow regulator coupled aport in said vessel; and a tubular member associated with said fluidflow regulator.
 22. An apparatus as recited in claim 21, wherein saidfluid flow regulator of said vessel comprises a valve selected from thegroup of valves consisting essentially of a stopcock, a check valve, aball valve, and a pressure release valve.
 23. An apparatus as recited inclaim 18, wherein said means for freezing said hyperpolarized noble gascomprises a cryogenic bath of liquid gases.
 24. An apparatus as recitedin claim 18, wherein said means for freezing said hyperpolarized noblegas comprises a refrigeration unit.
 25. An apparatus as recited in claim18, wherein said means for applying a magnetic field to said reservoircomprises a magnet selected from the group of magnets consistingessentially of a permanent magnet, an electromagnet, a superconductingelectromagnet, and a shimmed NMR spectrometer.